bims-tremyl Biomed News
on Therapy resistance biology in myeloid leukemia
Issue of 2024‒06‒23
forty-four papers selected by
Paolo Gallipoli, Barts Cancer Institute, Queen Mary University of London

  1. Leukemia. 2024 Jun 18.
      Chromosomal translocations of the nucleoporin 98 (NUP98) gene are found in acute myeloid leukemia (AML) patients leading to very poor outcomes. The oncogenic activity of NUP98 fusion proteins is dependent on the interaction between Mixed Lineage Leukemia 1 and menin. NUP98-rearranged (NUP98-r) leukemia cells also rely on specific kinases, including CDK6 and/or FLT3, suggesting that simultaneous targeting of these kinases and menin could overcome limited sensitivity to single agents. Here, we found that combinations of menin inhibitor, MI-3454, with kinase inhibitors targeting either CDK6 (Palbociclib) or FLT3 (Gilteritinib) strongly enhance the anti-leukemic effect of menin inhibition in NUP98-r leukemia models. We found strong synergistic effects of both combinations on cell growth, colony formation and differentiation in patient samples with NUP98 translocations. These combinations also markedly augmented anti-leukemic efficacy of menin inhibitor in Patient Derived Xenograft models of NUP98-r leukemia. Despite inhibiting two unrelated kinases, when Palbociclib or Gilteritinib were combined with the menin inhibitor, they affected similar pathways relevant to leukemogenesis, including cell cycle regulation, cell proliferation and differentiation. This study provides strong rationale for clinical translation of the combination of menin and kinase inhibitors as novel treatments for NUP98-r leukemia, supporting the unexplored combinations of epigenetic drugs with kinase inhibitors.
  2. Leukemia. 2024 Jun 18.
      Measurable residual disease (MRD) surveillance in acute myeloid leukemia (AML) may identify patients destined for relapse and thus provide the option of pre-emptive therapy to improve their outcome. Whilst flow cytometric MRD (Flow-MRD) can be applied to high-risk AML/ myelodysplasia patients, its diagnostic performance for detecting impending relapse is unknown. We evaluated this in a cohort comprising 136 true positives (bone marrows preceding relapse by a median of 2.45 months) and 155 true negatives (bone marrows during sustained remission). At an optimal Flow-MRD threshold of 0.040%, clinical sensitivity and specificity for relapse was 74% and 87% respectively (51% and 98% for Flow-MRD ≥ 0.1%) by 'different-from-normal' analysis. Median relapse kinetics were 0.78 log10/month but significantly higher at 0.92 log10/month for FLT3-mutated AML. Computational (unsupervised) Flow-MRD (C-Flow-MRD) generated optimal MRD thresholds of 0.036% and 0.082% with equivalent clinical sensitivity to standard analysis. C-Flow-MRD-identified aberrancies in HLADRlow or CD34+CD38low (LSC-type) subpopulations contributed the greatest clinical accuracy (56% sensitivity, 90% specificity) and notably, by longitudinal profiling expanded rapidly within blasts in > 40% of 86 paired MRD and relapse samples. In conclusion, flow MRD surveillance can detect MRD relapse in high risk AML and its evaluation may be enhanced by computational analysis.
  3. Blood. 2024 Jun 21. pii: blood.2023022480. [Epub ahead of print]
      The interaction between menin and histone-lysine N-methyltransferase 2A (KMT2A) is a critical dependency for KMT2A- or nucleophosmin 1 (NPM1)-altered leukemias and an emerging opportunity for therapeutic development. JNJ-75276617 is a novel, orally bioavailable, potent, and selective protein-protein interaction inhibitor of the binding between menin and KMT2A. In KMT2A-rearranged (KMT2A-r) and NPM1-mutant (NPM1c) AML cells, JNJ-75276617 inhibited the association of the menin-KMT2A complex with chromatin at target gene promoters, resulting in reduced expression of several menin-KMT2A target genes, including MEIS1 and FLT3. JNJ-75276617 displayed potent anti-proliferative activity across several AML and ALL cell lines and patient samples harboring KMT2A- or NPM1-alterations in vitro. In xenograft models of AML and ALL, JNJ-75276617 reduced leukemic burden and provided a significant dose-dependent survival benefit accompanied by expression changes of menin-KMT2A target genes. JNJ-75276617 demonstrated synergistic effects with gilteritinib in vitro in AML cells harboring KMT2A-r. JNJ-75276617 further exhibited synergistic effects with venetoclax and azacitidine in AML cells bearing KMT2A-r in vitro, and significantly increased survival in mice. Interestingly, JNJ-75276617 showed potent anti-proliferative activity in cell lines engineered with recently discovered mutations (MEN1M327I or MEN1T349M) that developed in patients refractory to the menin-KMT2A inhibitor revumenib. A co-crystal structure of menin in complex with JNJ-75276617 indicates a unique binding mode distinct from other menin-KMT2A inhibitors, including revumenib. JNJ-75276617 is being clinically investigated for acute leukemias harboring KMT2A or NPM1 alterations, as a monotherapy for relapsed/refractory (R/R) acute leukemia (NCT04811560), or in combination with AML-directed therapies (NCT05453903).
  4. Cancer. 2024 Jun 19.
      INTRODUCTION: NPM1-mutated (NPM1mut) myeloid neoplasms (MNs) with <20% bone marrow (BM) blasts (NPM1mut MNs<20) are uncommon, and their classification remains inconsistent.METHODS: The clinicopathologic features of 54 patients with NPM1mut MNs <20 were evaluated and compared with wild-type NPM1 MNs <20 and NPM1mut MNs≥20, respectively.
    RESULTS: NPM1mut MNs had similar features regardless of blast percentage, except for higher IDH2 (29% vs 7%, p = .023) and FLT3 (70% vs 11%, p < .001) frequency in patients with ≥20% BM blasts. Thirty-three (61%) patients with NPM1mut MNs <20 received low-intensity chemotherapy (LIC) and 12 (22%) received intensive chemotherapy (IC). Higher complete remission rates (75% vs 27%, p = .006) and median overall survival (mOS) (not reached vs 30.4 months, p = .06) were observed with IC compared to LIC. Young patients (age <60 years) did not reach mOS either when treated with LIC or IC. Stem cell transplant was associated with increased survival only in patients treated with LIC (HR, 0.24; p = .025). No differences in mOS were observed by BM blast strata (32.2 months, not reached and 46.9 months for <10%, 10%-19%, and ≥20% blasts, p = .700) regardless of treatment modality (LIC: p = .900; IC: p = .360). Twenty-three patients (43%) with NPM1mut MNs <20 had marrow blast progression to ≥20%.
    CONCLUSIONS: Overall, NPM1mut MNs define a unique entity independent of BM blast percentage.
    Keywords:  NPM1 mutation; intensive chemotherapy; myelodysplastic syndromes; myeloid neoplasms; stem cell transplant
  5. Ther Adv Hematol. 2024 ;15 20406207241257904
      Background: TP53 mutations are associated with an adverse prognosis in acute myeloid leukemia (AML) and higher-risk myelodysplastic syndromes (HR-MDS). However, the integrated genetic, epigenetic, and immunologic landscape of TP53-mutated AML/HR-MDS is not well defined.Objectives: To define the genetic, epigenetic, and immunologic landscape of TP53-mutant and TP53 wild-type AML and HR-MDS patients.
    Design: Post hoc analysis of TP53-mutant and TP53 wild-type patients treated on the randomized FUSION trial with azacitidine ± the anti-PD-L1 antibody durvalumab.
    Methods: We performed extensive molecular, epigenetic, and immunologic assays on a well-annotated clinical trial dataset of 61 patients with TP53-mutated disease (37 AML, 24 MDS) and 144 TP53 wild-type (89 AML, 55 MDS) patients, all of whom received azacitidine-based therapy. A 38 gene-targeted myeloid mutation analysis from screening bone marrow (BM) was performed. DNA methylation arrays, immunophenotyping and immune checkpoint expression by flow cytometry, and gene expression profiles by bulk RNA sequencing were assessed at baseline and serially during the trial.
    Results: Global DNA methylation from peripheral blood was independent of TP53 mutation and allelic status. AZA therapy led to a statistically significant decrease in global DNA methylation scores independent of TP53 mutation status. In BM from TP53-mutant patients, we found both a higher T-cell population and upregulation of inhibitory immune checkpoint proteins such as PD-L1 compared to TP53 wild-type. RNA sequencing analyses revealed higher expression of the myeloid immune checkpoint gene LILRB3 in TP53-mutant samples suggesting a novel therapeutic target.
    Conclusion: This integrated analysis of the genetic, epigenetic, and immunophenotypic landscape of TP53 mutant AML/HR-MDS suggests that differences in the immune landscape resulting in an immunosuppressive microenvironment rather than epigenetic differences contribute to the poor prognosis of TP53-mutant AML/HR-MDS with mono- or multihit TP53 mutation status.
    Trial registration: FUSION trial (NCT02775903).
    Keywords:  AML; MDS; TP53 mutation; gene expression; immune phenotype
  6. Br J Haematol. 2024 Jun 15.
      MLL-rearranged (MLL-r) leukaemia is observed in approximately 10% of acute myeloid leukaemia (AML) and is associated with a relatively poor prognosis, highlighting the need for new treatment regimens. MLL fusion proteins produced by MLL rearrangements recruit KDM4C to mediate epigenetic reprogramming, which is required for the maintenance of MLL-r leukaemia. In this study, we used a combinatorial drug screen to selectively identify synergistic treatment partners for the KDM4C inhibitor SD70. The results showed that the drug combination of SD70 and MI-503, a potent menin-MLL inhibitor, induced synergistically enhanced apoptosis in MLL::AF9 leukaemia cells without affecting normal CD34+ cells. In vivo treatment with SD70 and MI-503 significantly prolonged survival in AML xenograft models. Differential gene expression analysis by RNA-seq following combined pharmacological inhibition of SD70 and MI-503 revealed changes in numerous genes, with MYC target genes being the most significantly downregulated. Taken together, these data provide preclinical evidence that the combination of SD70 and MI-503 is a potential dual-targeted therapy for MLL::AF9 AML.
    Keywords:   MLL::AF9 ; AML; MI‐503; SD70; combination therapy
  7. Br J Haematol. 2024 Jun 12.
      Upregulation of the Wilms' tumour 1 (WT1) gene is common in acute myeloid leukaemia (AML) and is associated with poor prognosis. WT1 generates 12 primary transcripts through different translation initiation sites and alternative splicing. The short WT1 transcripts express abundantly in primary leukaemia samples. We observed that overexpression of short WT1 transcripts lacking exon 5 with and without the KTS motif (sWT1+/- and sWT1-/-) led to reduced cell growth. However, only sWT1+/- overexpression resulted in decreased CD71 expression, G1 arrest, and cytarabine resistance. Primary AML patient cells with low CD71 expression exhibit resistance to cytarabine, suggesting that CD71 may serve as a potential biomarker for chemotherapy. RNAseq differential expressed gene analysis identified two transcription factors, HOXA3 and GATA2, that are specifically upregulated in sWT1+/- cells, whereas CDKN1A is upregulated in sWT1-/- cells. Overexpression of either HOXA3 or GATA2 reproduced the effects of sWT1+/-, including decreased cell growth, G1 arrest, reduced CD71 expression and cytarabine resistance. HOXA3 expression correlates with chemotherapy response and overall survival in NPM1 mutation-negative leukaemia specimens. Overexpression of HOXA3 leads to drug resistance against a broad spectrum of chemotherapeutic agents. Our results suggest that WT1 regulates cell proliferation and drug sensitivity in an isoform-specific manner.
    Keywords:  HOXA3; WT1; biomarkers; chemotherapy resistance
  8. Sci Transl Med. 2024 Jun 12. 16(751): eadi5336
      In chronic myeloid leukemia (CML), the persistence of leukemic stem cells (LSCs) after treatment with tyrosine kinase inhibitors (TKIs), such as imatinib, can lead to disease relapse. It is known that therapy-resistant LSCs rely on oxidative phosphorylation (OXPHOS) for their survival and that targeting mitochondrial respiration sensitizes CML LSCs to imatinib treatment. However, current OXPHOS inhibitors have demonstrated limited efficacy or have shown adverse effects in clinical trials, highlighting that identification of clinically safe oxidative pathway inhibitors is warranted. We performed a high-throughput drug repurposing screen designed to identify mitochondrial metabolism inhibitors in myeloid leukemia cells. This identified lomerizine, a US Food and Drug Administration (FDA)-approved voltage-gated Ca2+ channel blocker now used for the treatment of migraines, as one of the top hits. Transcriptome analysis revealed increased expression of voltage-gated CACNA1D and receptor-activated TRPC6 Ca2+ channels in CML LSCs (CD34+CD38-) compared with normal counterparts. This correlated with increased endoplasmic reticulum (ER) mass and increased ER and mitochondrial Ca2+ content in CML stem/progenitor cells. We demonstrate that lomerizine-mediated inhibition of Ca2+ uptake leads to ER and mitochondrial Ca2+ depletion, with similar effects seen after CACNA1D and TRPC6 knockdown. Through stable isotope-assisted metabolomics and functional assays, we observe that lomerizine treatment inhibits mitochondrial isocitrate dehydrogenase activity and mitochondrial oxidative metabolism and selectively sensitizes CML LSCs to imatinib treatment. In addition, combination treatment with imatinib and lomerizine reduced CML tumor burden, targeted CML LSCs, and extended survival in xenotransplantation model of human CML, suggesting this as a potential therapeutic strategy to prevent disease relapse in patients.
  9. Cardiovasc Hematol Disord Drug Targets. 2024 Jun 11.
      The development of myeloid malignancies is a multi-step process starting from pre-malignant stages. Large-scale studies on clonal hematopoiesis of indeterminate potential (CHIP) identified this condition as a risk factor for developing hematologic malignancies, in particular myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). In parallel, CHIP was found to confer an enhanced thrombotic risk, in particular for cardiovascular diseases. In a similar fashion, in recent years, alongside their life-threatening features, increasing attention has been drawn toward thrombotic complications in myeloid malignancies. The purpose of this review is to gather growing body of evidence on incidence, pathogenesis and clinical impact of thrombosis in myeloid malignancies at every step of malignant progression, from CHIP to AML.
    Keywords:  Acute Myeloid Leukemia; Acute Promyelocytic Leukemia; Clonal Hemopoiesis of indeterminate potential; Myelodysplastic Syndromes; Thrombosis; VEXAS Syndrome
  10. Int J Mol Sci. 2024 May 30. pii: 6020. [Epub ahead of print]25(11):
      Pediatric acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) exhibit favorable survival rates. However, for AML and ALL patients carrying KMT2A gene translocations clinical outcome remains unsatisfactory. Key players in KMT2A-fusion-driven leukemogenesis include menin and DOT1L. Recently, menin inhibitors like revumenib have garnered attention for their potential therapeutic efficacy in treating KMT2A-rearranged acute leukemias. However, resistance to menin inhibition poses challenges, and identifying which patients would benefit from revumenib treatment is crucial. Here, we investigated the in vitro response to revumenib in KMT2A-rearranged ALL and AML. While ALL samples show rapid, dose-dependent induction of leukemic cell death, AML responses are much slower and promote myeloid differentiation. Furthermore, we reveal that acquired resistance to revumenib in KMT2A-rearranged ALL cells can occur either through the acquisition of MEN1 mutations or independently of mutations in MEN1. Finally, we demonstrate significant synergy between revumenib and the DOT1L inhibitor pinometostat in KMT2A-rearranged ALL, suggesting that such drug combinations represent a potent therapeutic strategy for these patients. Collectively, our findings underscore the complexity of resistance mechanisms and advocate for precise patient stratification to optimize the use of menin inhibitors in KMT2A-rearranged acute leukemia.
    Keywords:  ALL; AML; DOT1L; KMT2A-rearranged; infant; menin; pediatric; pinometostat; revumenib; synergy
  11. Ann Hematol. 2024 Jun 17.
    PETHEMA group
      FLT3-ITD and NPM1 mutations are key to defining the genetic risk profile of acute myeloid leukemia (AML). We aimed to assess the prognostic features of the FLT3-ITD and NPM1 mutations in old and/or unfit individuals with AML treated with non-intensive therapies in the era before azacitidine-venetoclax approbation. The results of various non-intensive regimens were also compared. We conducted a retrospective analysis that included patients treated with different non-intensive regimens, between 2007 and 2020 from PETHEMA AML registry. We compiled 707 patients with a median age of 74 years and median follow-up time of 37.7 months. FLT3-ITD patients (N = 98) showed a non-significant difference in overall survival (OS) compared to FLT3-ITD negative-patients (N = 608) (P = 0.17, median OS was 5 vs 7.3 months respectively). NPM1-mutated patients (N = 144) also showed a non-significant difference with NPM1 wild type (N = 519) patients (P = 0.25, median OS 7.2 vs 6.8 respectively). In the Cox regression analysis neither NPM1 nor FLT3-ITD nor age were significant prognostic variables for OS prediction. Abnormal karyotype and a high leukocyte count showed a statistically significant deleterious effect. Azacitidine also showed better survival compared to FLUGA (low dose cytarabine plus fludarabine). NPM1 and FLT3-ITD seem to lack prognostic value in older/unfit AML patients treated with non-intensive regimens other than azacitidine-venetoclax combination.
    Keywords:  Acute myeloid leukemia; Elderly; Fms-like tyrosine kinase 3 (FLT3) mutation; Hypomethylating; Nucleophosmin 1 (NPM1) mutation; PETHEMA; Treatment
  12. Elife. 2024 Jun 18. pii: RP91611. [Epub ahead of print]12
      The DNA damage response is critical for maintaining genome integrity and is commonly disrupted in the development of cancer. PPM1D (protein phosphatase Mg2+/Mn2+-dependent 1D) is a master negative regulator of the response; gain-of-function mutations and amplifications of PPM1D are found across several human cancers making it a relevant pharmacological target. Here, we used CRISPR/Cas9 screening to identify synthetic-lethal dependencies of PPM1D, uncovering superoxide dismutase-1 (SOD1) as a potential target for PPM1D-mutant cells. We revealed a dysregulated redox landscape characterized by elevated levels of reactive oxygen species and a compromised response to oxidative stress in PPM1D-mutant cells. Altogether, our results demonstrate a role for SOD1 in the survival of PPM1D-mutant leukemia cells and highlight a new potential therapeutic strategy against PPM1D-mutant cancers.
    Keywords:  DNA damage; cancer; cancer biology; cell biology; leukemia; mouse
  13. Hemasphere. 2024 Jun;8(6): e90
      Transcriptional cofactors of the ETO family are recurrent fusion partners in acute leukemia. We characterized the ETO2 regulome by integrating transcriptomic and chromatin binding analyses in human erythroleukemia xenografts and controlled ETO2 depletion models. We demonstrate that beyond its well-established repressive activity, ETO2 directly activates transcription of MYB, among other genes. The ETO2-activated signature is associated with a poorer prognosis in erythroleukemia but also in other acute myeloid and lymphoid leukemia subtypes. Mechanistically, ETO2 colocalizes with EP300 and MYB at enhancers supporting the existence of an ETO2/MYB feedforward transcription activation loop (e.g., on MYB itself). Both small-molecule and PROTAC-mediated inhibition of EP300 acetyltransferases strongly reduced ETO2 protein, chromatin binding, and ETO2-activated transcripts. Taken together, our data show that ETO2 positively enforces a leukemia maintenance program that is mediated in part by the MYB transcription factor and that relies on acetyltransferase cofactors to stabilize ETO2 scaffolding activity.
  14. Hum Pathol. 2024 Jun 13. pii: S0046-8177(24)00109-6. [Epub ahead of print]
      CSF3R activating mutation is a genetic hallmark of chronic neutrophilic leukemia (CNL), and is also present in a subset of atypical chronic myeloid leukemia (aCML), but infrequent in other myeloid neoplasms. However, the occurrence of CSF3R mutations in various myeloid neoplasms is not well studied. Here we evaluate the spectrum of CSF3R mutations and the clinicopathologic features of CSF3R mutated myeloid neoplasms. We retrospectively identified CSF3R mutations in a variety of myeloid neoplasms: two CNL, three atypical chronic myeloid leukemia (aCML), nine acute myeloid leukemia (AML), one chronic myelomonocytic leukemia, and one myeloproliferative neoplasm. The prototypic T618I mutation was found in 50% of cases: CNL (2/2), aCML (2/3) and AML (4/9). We observed a new recurrent CSF3R mutation Q776* in 25% of cases, and a potential-germline mutation in a 20-year-old patient. Co-occurring mutations were often in epigenetic modifier and spliceosome. IDH/RUNX1 and tumor suppressor mutations were frequent in AML but absent in CNL/aCML. All CNL/aCML patients succumbed within 2-years of diagnosis. We demonstrate that CSF3R mutations are not restricted to CNL. CNL and aCML show similar clinicopathologic and molecular features, suggesting that CNL may be best classified as myelodysplastic/myeloproliferative neoplasm rather than myeloproliferative neoplasm.
    Keywords:  Atypical chronic myeloid leukemia (aCML); Chronic neutrophilic leukemia (CNL); Colony stimulating factor 3 receptor (CSF3R); Myeloid neoplasms
  15. Exp Hematol. 2024 Jun 13. pii: S0301-472X(24)00112-7. [Epub ahead of print] 104253
      Acute myeloid leukemias are a group of hematological malignancies characterized by a poor prognosis for survival. The discovery of oncogenic mutations in the FLT3 gene has led to the development of tyrosine kinase inhibitors such as Quizartinib. However, achieving complete remission in patients remains challenging because these new TKIs are unable to completely eradicate all leukemic cells. Residual leukemic cells persist during Quizartinib treatment, leading to the rapid emergence of drug-resistant leukemia. Given that mitochondrial oxidative metabolism promotes the survival of leukemic cells after exposure to multiple anticancer drugs, we characterized the metabolism of leukemic cells that persisted during Quizartinib treatment and developed metabolic strategies to eradicate them. In our study, employing biochemical and metabolomics approaches, we confirmed that the survival of leukemic cells treated with FLT3 inhibitors critically depends on maintaining mitochondrial metabolism, specifically through glutamine oxidation. We uncovered a synergistic interaction between the FLT3 inhibitor Quizartinib and L-Asparaginase, operating through anti-metabolic mechanisms. Utilizing various models of persistent leukemia, we demonstrated that leukemic cells resistant to Quizartinib are susceptible to L-Asparaginase. This combined therapeutic strategy shows promise in reducing the development of resistance to FLT3 inhibitors, offering a potential strategy to enhance treatment outcomes.
    Keywords:  AML; energy metabolism; glycolysis; leukemia; mitochondria; resistance
  16. Ann Hematol. 2024 Jun 15.
      Erdheim-Chester disease (ECD) is a rare histiocytosis that tends to co-exist with other myeloid malignancies. Here, we use genetic and transcriptomic sequencing to delineate a case of co-occurring BRAFV600E-mutated ECD and acute myeloid leukemia (AML), followed by AML remission and relapse. The AML relapse involved the extinction of clones with KMT2A-AFDN and FLT3-ITD, and the predominance of PTPN11-mutated subclones with distinct transcriptomic features. This case report has highlighted the screening for other myeloid malignancies at the diagnosis of ECD and the clinical significance of PTPN11-mutated AML subclones that require meticulous monitoring.
    Keywords:   FLT3-ITD; PTPN11 ; Acute myeloid leukemia; Erdheim-Chester disease
  17. Br J Haematol. 2024 Jun 18.
      The transcription factor GATA2 has a pivotal role in haematopoiesis. Heterozygous germline GATA2 mutations result in a syndrome characterized by immunodeficiency, bone marrow failure and predispositions to myelodysplastic syndrome (MDS) and acute myeloid leukaemia. Clinical symptoms in these patients are diverse and mechanisms driving GATA2-related phenotypes are largely unknown. To explore the impact of GATA2 haploinsufficiency on haematopoiesis, we generated a zebrafish model carrying a heterozygous mutation of gata2b (gata2b+/-), an orthologue of GATA2. Morphological analysis revealed myeloid and erythroid dysplasia in gata2b+/- kidney marrow. Because Gata2b could affect both transcription and chromatin accessibility during lineage differentiation, this was assessed by single-cell (sc) RNA-seq and single-nucleus (sn) ATAC-seq. Sn-ATAC-seq showed that the co-accessibility between the transcription start site (TSS) and a -3.5-4.1 kb putative enhancer was more robust in gata2b+/- zebrafish HSPCs compared to wild type, increasing gata2b expression and resulting in higher genome-wide Gata2b motif use in HSPCs. As a result of increased accessibility of the gata2b locus, gata2b+/- chromatin was also more accessible during lineage differentiation. scRNA-seq data revealed myeloid differentiation defects, that is, impaired cell cycle progression, reduced expression of cebpa and cebpb and increased signatures of ribosome biogenesis. These data also revealed a differentiation delay in erythroid progenitors, aberrant proliferative signatures and down-regulation of Gata1a, a master regulator of erythropoiesis, which worsened with age. These findings suggest that cell-intrinsic compensatory mechanisms, needed to obtain normal levels of Gata2b in heterozygous HSPCs to maintain their integrity, result in aberrant lineage differentiation, thereby representing a critical step in the predisposition to MDS.
    Keywords:  GATA2 deficiency syndrome; Gata2b; HSCs; MDS; epigenetic compensation; zebrafish
  18. Haematologica. 2024 Jun 20.
      Hematopoietic cell transplantation (HCT) is the only potentially curative treatment option for many patients with hematologic malignancies. While HCT outcomes have improved drastically over the years, patients and clinicians continue to face numerous survivorship challenges, such as relapse, graft-versushost disease, and secondary malignancies. Recent literature suggests that clonal hematopoiesis (CH), the presence of a recurrent somatic mutation in hematopoietic cells, in HCT patients or donors may be associated with outcomes in autologous and allogeneic HCT. Herein, we perform a review of the literature and summarize reported associations between CH and clinical outcomes in HCT. For commonly reported outcomes, we used meta-analysis methods to provide estimates of effect sizes when combining results. A total of 32 articles with relevant and independent contributions were included, covering both autologous (n = 19) and allogeneic (n = 13) HCT. The articles report variable risk for developing outcomes according to CH characteristics, patient disease status, and method of HCT. Using meta-analysis of available results, HCT outcomes with statistically significant effects by CH status include therapy-related myeloid neoplasms (OR 3.65, 95%CI 2.18-6.10) and overall survival (HR 1.38, 95%CI 1.20-1.58) in autologous HCT and relapse (HR 0.80, 95%CI 0.68-0.94) in allogeneic HCT. However, heterogeneity, biases, and limitations in the literature provide challenges for informing the translation of CH to clinical decision-making. We conclude with a call to action and discussion of next steps to build upon the current literature and provide granularity to the true clinical impact of CH in the setting of HCT.
  19. Blood. 2024 Jun 21. pii: blood.2023019986. [Epub ahead of print]
      The ribosomopathy Shwachman-Diamond syndrome (SDS) is a rare autosomal recessive inherited bone marrow failure syndrome (IBMFS) caused by mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene, that is associated with an increased risk of myeloid malignancy. Tracking how hematopoietic stem cell (HSC) clonal dynamics change over time, assessing whether somatic genetic rescue mechanisms affect these dynamics, and mapping out when leukemic driver mutations are acquired is important to understand which individuals with SDS may go on to develop leukemia. In this review, we will discuss how new technologies that allow researchers to map mutations at the level of single HSC clones are generating important insights into genetic rescue mechanisms and their relative risk for driving evolution to leukemia, and how these data can inform the future development of personalized medicine approaches in SDS and other IBMFSs.
  20. Am J Hematol. 2024 Jun 20.
      Venetoclax-azacitidine is the standard of treatment for unfit acute myeloid leukemia patients. In the VIALE-A study, treatment was given until progression but there are no data on its optimal duration for responding patients who do not tolerate indefinite therapy. We retrospectively analyzed the outcome of patients who discontinued venetoclax or venetoclax-azacitidine due to poor tolerance. Sixty-two newly diagnosed (ND) AML patients and 22 patients with morphological relapse or refractory AML were included. In the ND cohort (n = 62), 28 patients stopped venetoclax and azacitidine and 34 patients continued azacitidine monotherapy. With a median follow-up of 23 months (IQR, 20-32), median overall survival and treatment-free survival were 44 (IQR, 16-NR) and 16 (IQR, 8-27) months, respectively. Patients who stopped both treatments and those who continued azacitidine monotherapy had the same outcomes. Negative minimal residual disease was associated with a 2-year treatment-free survival of 80%. In the RR cohort (n = 22), median overall survival and treatment-free survival were 19 (IQR, 17-31) and 10 (IQR, 5-NR) months, respectively. Prior number of venetoclax-azacitidine cycles and IDH mutations were associated with increased overall survival. The only factor significantly impacting treatment-free survival was the number of prior cycles. This study suggests that patients who discontinued treatment in remission have favorable outcomes supporting the rationale for prospective controlled trials.
  21. Biomacromolecules. 2024 Jun 13.
      Acute myeloid leukemia (AML) is often associated with poor prognosis and survival. Small molecule inhibitors, though widening the treatment landscape, have limited monotherapy efficacy. The combination therapy, however, shows suboptimal clinical outcomes due to low bioavailability, overlapping systemic toxicity and drug resistance. Here, we report that CXCR4-mediated codelivery of the BCL-2 inhibitor venetoclax (VEN) and the FLT3 inhibitor sorafenib (SOR) via T22 peptide-tagged disulfide cross-linked polymeric micelles (TM) achieves synergistic treatment of FLT3-ITD AML. TM-VS with a VEN/SOR weight ratio of 1/4 and T22 peptide density of 20% exhibited an extraordinary inhibitory effect on CXCR4-overexpressing MV4-11 AML cells. TM-VS at a VEN/SOR dosage of 2.5/10 mg/kg remarkably reduced leukemia burden, prolonged mouse survival, and impeded bone loss in orthotopic MV4-11-bearing mice, outperforming the nontargeted M-VS and oral administration of free VEN/SOR. CXCR4-mediated codelivery of BCL-2 and FLT3 inhibitors has emerged as a prospective clinical treatment for FLT3-ITD AML.
  22. bioRxiv. 2024 Jun 09. pii: 2024.06.06.597775. [Epub ahead of print]
      Hematopoietic stem cells (HSCs) with multilineage potential are critical for effective T cell reconstitution and restoration of the adaptive immune system after allogeneic Hematopoietic Cell Transplantation (allo-HCT). Our current understanding of lymphoid-poised HSC subsets is limited due to a lack of reliable markers. Through comprehensive molecular profiling of HSCs, we identify Kit lo HSCs-a subset with enhanced lymphoid potential, which declines with age. Using a preclinical allo-HCT model, we demonstrate that Kit lo HSCs exhibit superior lymphoid progenitor output, enhanced thymic recovery, and T-cell reconstitution resulting in improved T cell responses to infection post-HCT. Furthermore, Kit lo HSCs with augmented BM lymphopoiesis mitigate age-associated thymic alterations, thus enhancing T-cell recovery in middle-aged hosts. Chromatin profiling revealed that Kit lo HSCs exhibit higher activity of lymphoid-specifying transcription factors (TFs), including Zbtb1 . Deletion of Zbtb1 in Kit lo HSCs, diminished their T-cell potential, while reinstating Zbtb1 in megakaryocytic-biased Kit hi HSCs rescued T-cell potential, in vitro and in vivo . Finally, we discover an analogous Kit lo HSC subset with enhanced lymphoid potential in human bone marrow. Our results demonstrate that Kit lo HSCs identify HSC subsets with enhanced lymphoid potential mediated by an underlying epigenetic program.
  23. Int J Mol Sci. 2024 Jun 06. pii: 6256. [Epub ahead of print]25(11):
      Acute Erythroid Leukemia (AEL) is a rare and aggressive subtype of Acute Myeloid Leukemia (AML). In 2022, the World Health Organization (WHO) defined AEL as a biopsy with ≥30% proerythroblasts and erythroid precursors that account for ≥80% of cellularity. The International Consensus Classification refers to this neoplasm as "AML with mutated TP53". Classification entails ≥20% blasts in blood or bone marrow biopsy and a somatic TP53 mutation (VAF > 10%). This type of leukemia is typically associated with biallelic TP53 mutations and a complex karyotype, specifically 5q and 7q deletions. Transgenic mouse models have implicated several molecules in the pathogenesis of AEL, including transcriptional master regulator GATA1 (involved in erythroid differentiation), master oncogenes, and CDX4. Recent studies have also characterized AEL by epigenetic regulator mutations and transcriptome subgroups. AEL patients have overall poor clinical outcomes, mostly related to their poor response to the standard therapies, which include hypomethylating agents and intensive chemotherapy. Allogeneic bone marrow transplantation (AlloBMT) is the only potentially curative approach but requires deep remission, which is very challenging for these patients. Age, AlloBMT, and a history of antecedent myeloid neoplasms further affect the outcomes of these patients. In this review, we will summarize the diagnostic criteria of AEL, review the current insights into the biology of AEL, and describe the treatment options and outcomes of patients with this disease.
    Keywords:  Acute Erythroid Leukemia; Acute Myeloid Leukemia; hematopoietic disorders; myeloid neoplasms
  24. EJHaem. 2024 Jun;5(3): 560-564
      Relapse remains a major cause of treatment failure following allogeneic stem cell transplantation (allo-SCT) for patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We retrospectively investigated low-dose decitabine and venetoclax (DEC/VEN) as post-transplant maintenance in 26 older patients with AML and MDS. The cumulative incidence of day 100 gIII-IV acute graft versus host disease (GVHD) and 1-year moderate-severe chronic GVHD was 5% and 26%, respectively. One patient relapsed 14 m after transplant. The 1-year non-relapse mortality and survival were 11% and 84%, respectively. DEC/VEN is a safe and potentially effective strategy to reduce the risk of post-transplant relapse.
    Keywords:  decitabine; maintenance chemotherapy; myeloid leukemia; post‐transplant; venetoclax
  25. EJHaem. 2024 Jun;5(3): 565-572
      The PICALM::MLLT10 fusion gene is a rare but recurrent event in acute leukemia (AL) associated with poor prognosis. It is still confused whether PICALM::MLLT10 can solely correspond to acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) or acute leukemias of ambiguous lineage (ALAL). Here, we reported a series of PICALM::MLLT10 positive AL patients with miscellaneous immunophenotype including T-ALL, ALAL, AML, and B-ALL, complex karyotype, half of extramedullary disease (EMD), frequently concomitant PHF6 mutation, and poor initial treatment response to standard chemotherapy aiming to different immunophenotype, but showing sensitivity to combining chemotherapy especially integrated with venetoclax, suggesting this fusion gene may indicate a new subgroup of AL. Eighteen PICALM::MLLT10 positive patients of 533 AL patients (18/533, 3.4%) were identified by RNA sequencing in our center. We found PICALM::MLLT10 positive AL showing miscellaneous immunophenotype, higher expression of leukemic stemness genes and lower expression of biomarkers of venetoclax resistance, more extramedullary involvement, and especially poor response to conventional induction chemotherapy, but may benefit from venetoclax as well as low-dose Ara-C, granulocyte colony-stimulating factor (G-CSF), and anthracyclines combination chemotherapy. Sequential hematopoietic stem cell transplantation (HSCT) after chemotherapy combined with venetoclax may further improve long-term survival in AL patients with complete remission (CR) even measurable residual disease (MRD) positive.
    Keywords:  PICALM::MLLT10; acute leukemia; immunophenotyping; venetoclax
  26. Blood Cancer J. 2024 Jun 18. 14(1): 99
      Current therapies for high-grade TP53-mutated myeloid neoplasms (≥10% blasts) do not offer a meaningful survival benefit except allogeneic stem cell transplantation in the minority who achieve a complete response to first line therapy (CR1). To identify reliable pre-therapy predictors of complete response to first-line therapy (CR1) and outcomes, we assembled a cohort of 242 individuals with TP53-mutated myeloid neoplasms and ≥10% blasts with well-annotated clinical, molecular and pathology data. Key outcomes examined were CR1 & 24-month survival (OS24). In this elderly cohort (median age 68.2 years) with 74.0% receiving frontline non-intensive regimens (hypomethylating agents +/- venetoclax), the overall cohort CR1 rate was 25.6% (50/195). We additionally identified several pre-therapy factors predictive of inferior CR1 including male gender (P = 0.026), ≥2 autosomal monosomies (P < 0.001), -17/17p (P = 0.011), multi-hit TP53 allelic state (P < 0.001) and CUX1 co-alterations (P = 0.010). In univariable analysis of the entire cohort, inferior OS24 was predicated by ≥2 monosomies (P = 0.004), TP53 VAF > 25% (P = 0.002), TP53 splice junction mutations (P = 0.007) and antecedent treated myeloid neoplasm (P = 0.001). In addition, mutations/deletions in CUX1, U2AF1, EZH2, TET2, CBL, or KRAS ('EPI6' signature) predicted inferior OS24 (HR = 2.0 [1.5-2.8]; P < 0.0001). In a subgroup analysis of HMA +/-Ven treated individuals (N = 144), TP53 VAF and monosomies did not impact OS24. A risk score for HMA +/-Ven treated individuals incorporating three pre-therapy predictors including TP53 splice junction mutations, EPI6 and antecedent treated myeloid neoplasm stratified 3 prognostic distinct groups: intermediate, intermediate-poor, and poor with significantly different median (12.8, 6.0, 4.3 months) and 24-month (20.9%, 5.7%, 0.5%) survival (P < 0.0001). For the first time, in a seemingly monolithic high-risk cohort, our data identifies several baseline factors that predict response and 24-month survival.
  27. Proc Natl Acad Sci U S A. 2024 Jun 25. 121(26): e2405905121
      Aberrant regulation of chromatin modifiers is a common occurrence across many cancer types, and a key priority is to determine how specific alterations of these proteins, often enzymes, can be targeted therapeutically. MOZ, a histone acyltransferase, is recurrently fused to coactivators CBP, p300, and TIF2 in cases of acute myeloid leukemia (AML). Using either pharmacological inhibition or targeted protein degradation in a mouse model for MOZ-TIF2-driven leukemia, we show that KAT6 (MOZ/MORF) enzymatic activity and the MOZ-TIF2 protein are necessary for indefinite proliferation in cell culture. MOZ-TIF2 directly regulates a small subset of genes encoding developmental transcription factors, augmenting their high expression. Furthermore, transcription levels in MOZ-TIF2 cells positively correlate with enrichment of histone H3 propionylation at lysine 23 (H3K23pr), a recently appreciated histone acylation associated with gene activation. Unexpectedly, we also show that MOZ-TIF2 and MLL-AF9 regulate transcription of unique gene sets, and their cellular models exhibit distinct sensitivities to multiple small-molecule inhibitors directed against AML pathways. This is despite the shared genetic pathways of wild-type MOZ and MLL. Overall, our data provide insight into how aberrant regulation of MOZ contributes to leukemogenesis. We anticipate that these experiments will inform future work identifying targeted therapies in the treatment of AML and other diseases involving MOZ-induced transcriptional dysregulation.
    Keywords:  AML; KAT6; MOZ; propionylation
  28. Leuk Lymphoma. 2024 Jun 17. 1-7
      Recent updates in the classification of myeloid neoplasms (MNs) recognize the poor prognostic impact of TP53 mutations, with particular emphasis on the TP53 allele status. Studies on the effect of TP53 allele status exclusively in therapy-related MNs (t-MNs) are lacking. We compared the clinicopathologic and survival characteristics of t-MNs with single-hit (SH) and multi-hit (MH) TP53 mutations. A total of 71 TP53-mutated t-MNs were included, including 56 (78.9%) MH and 15 (21.1%) SH. Both groups showed comparable genetic profiles with an excess of high-risk karyotypes and a paucity of other co-mutated genes. TP53 was the sole detectable mutation in 73.3% of SH and 75.0% of MH cases. The overall survival (OS) of SH TP53-mutated t-MNs was not significantly different from MH cases (median survival: 233 vs.273 days, p = 0.70). Our findings suggest that t-MNs with SH TP53 mutations share the poor prognostic and biologic profile of their MH counterparts.
    Keywords:  T-MN; TP53; biallelic; monoallelic; multi-hit; single-hit
  29. Res Sq. 2024 Jun 03. pii: [Epub ahead of print]
      Mutations in RNA splicing factor genes including SF3B1, U2AF1, SRSF2, and ZRSR2 have been reported to contribute to development of myeloid neoplasms including myelodysplastic syndrome (MDS) and secondary acute myeloid leukemia (sAML). Chemical tools targeting cells carrying these mutant genes remain limited and underdeveloped. Among the four proteins, mutant U2AF1 (U2AF1 mut ) acquires an altered 3' splice site selection preference and co-operates with the wild-type U2AF1 (U2AF1 wt ) to change various gene isoform patterns to support MDS cells survival and proliferation. U2AF1 mutations in MDS cells are always heterozygous and the cell viability is reduced when exposed to additional insult affecting U2AF1 wt function. To investigate if the pharmacological inhibition of U2AF1wt function can provoke drug-induced vulnerability of cells harboring U2AF1mut, we conducted a fragment-based library screening campaign to discover compounds targeting the U2AF homology domain (UHM) in U2AF1 that is required for the formation of the U2AF1/U2AF2 complex to define the 3' splice site. The most promising hit (SF1-8) selectively inhibited growth of leukemia cell lines overexpressingU2AF1 mut and human primary MDS cells carrying U2AF1 mut . RNA-seq analysis of K562-U2AF1 mut following treatment with SF1-8 further revealed alteration of isoform patterns for a set of proteins that impair or rescue pathways associated with endocytosis, intracellular vesicle transport, and secretion. Our data suggested that further optimization of SF1-8 is warranted to obtain chemical probes that can be used to evaluate the therapeutic concept of inducing lethality to U2AF1 mut cells by inhibiting the U2AF1 wt protein.
  30. Cancer Res. 2024 Jun 17.
      Increasing evidence supports the interplay between oncogenic mutations and immune escape mechanisms. Strategies to counteract the immune escape mediated by oncogenic signaling could provide improved therapeutic options for patients with various malignancies. As mutant calreticulin (CALR) is a common driver of myeloproliferative neoplasms (MPN), we analyzed the impact of oncogenic CALRdel52 on the bone marrow (BM) microenvironment in MPN. Single-cell RNA-sequencing revealed that CALRdel52 led to the expansion of TGF-β1-producing erythroid progenitor cells and promoted the expansion of FoxP3+ regulatory T cells (Treg) in a murine MPN model. Treatment with an anti-TGF-β antibody improved mouse survival and increased the glycolytic activity in CD4+ and CD8+ T cells in vivo, while T cell depletion abrogated the protective effects conferred by neutralizing TGF-β. TGF-β1 reduced perforin and TNF-α production by T cells in vitro. TGF-β1 production by CALRdel52 cells was dependent on JAK1/2, PI3K, and ERK activity, which activated the transcription factor Sp1 to induce TGF-β1 expression. In four independent patient cohorts, TGF-β1 expression was increased in the BM of MPN patients compared to healthy individuals, and the BM of MPN patients contained a higher frequency of Treg compared to healthy individuals. Together, this study identified an ERK/Sp1/TGF-β1 axis in CALRdel52 MPNs as a mechanism of immunosuppression that can be targeted to elicit T-cell-mediated cytotoxicity.
  31. bioRxiv. 2024 Jun 08. pii: 2024.06.07.596542. [Epub ahead of print]
      A defined number of hematopoietic stem cell (HSC) clones are born during development and expand to form the pool of adult stem cells. An intricate balance between self-renewal and differentiation of these HSCs supports hematopoiesis for life. HSC fate is determined by complex transcription factor networks that drive cell-type specific gene programs. The transcription factor RUNX1 is required for definitive hematopoiesis, and mutations in Runx1 have been shown to reduce clonal diversity. The RUNX1 cofactor, CBFý, stabilizes RUNX1 binding to DNA, and disruption of their interaction alters downstream gene expression. Chemical screening for modulators of Runx1 and HSC expansion in zebrafish led us to identify a new mechanism for the RUNX1 inhibitor, Ro5-3335. We found that Ro5-3335 increased HSC divisions in zebrafish, and animals transplanted with Ro5-3335 treated cells had enhanced chimerism compared to untreated cells. Using human CD34+ cells, we show that Ro5-3335 remodels the RUNX1 transcription complex by binding to ELF1, independent of CBFý. This allows specific expression of cell cycle and hematopoietic genes that enhance HSC self-renewal and prevent differentiation. Furthermore, we provide the first evidence to show that it is possible to pharmacologically increase the number of stem cell clones in vivo , revealing a previously unknown mechanism for enhancing clonal diversity. Our studies have revealed a mechanism by which binding partners of RUNX1 determine cell fate, with ELF transcription factors guiding cell division. This information could lead to treatments that enhance clonal diversity for blood diseases.
  32. Ann Hematol. 2024 Jun 18.
      Here, we present a rare case of myeloproliferative neoplasms (MPN) with eosinophilia harboring both BCR::ABL1 and PDGFRB rearrangements, posing a classification dilemma. The patient exhibited clinical and laboratory features suggestive of chronic myeloid leukemia (CML) and myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions (MLN-TK), highlighting the diagnostic challenges associated with overlapping phenotypes. Despite the complexity, imatinib treatment swiftly achieved deep molecular remission, underscoring the therapeutic efficacy of tyrosine kinase inhibitors in such scenarios. Furthermore, the rapid attainment of deep remission by this patient in response to imatinib closely resembles that observed in MLN-TK patients with PDGFRB rearrangements. Further research is warranted to elucidate the underlying mechanisms driving the coexistence of multiple oncogenic rearrangements in MPNs and to optimize therapeutic strategies for these complex cases.
    Keywords:  BCR:ABL1; CML; MLN-TK; MPN; PDGFRB
  33. Am J Hematol. 2024 Jun 17.
      In patients with lower-risk myelodysplastic syndromes/neoplasms (MDS), response to first-line therapy is limited and transient. The MATTERHORN randomized, double-blind, phase 3 trial evaluated roxadustat versus placebo for patients with transfusion-dependent, lower-risk MDS. Eligible patients had very low-, low-, or intermediate-risk MDS with or without prior erythropoiesis-stimulating agent treatment, and a transfusion burden of 1-4 packed red blood cell (pRBC) units every 8 weeks (Q8W). Patients were randomized (3:2) to oral roxadustat (2.5 mg/kg) or placebo, both three times weekly, with best supportive care. Primary efficacy endpoint was transfusion independence (TI) for ≥56 days within 28 weeks (TI responders). MATTERHORN was terminated due to interim analysis outcomes not meeting statistical significance. In total, 272 patients were screened, and 140 patients were enrolled (82, roxadustat, and 58, placebo). At final analysis, 38/80 (47.5%) patients and 19/57 (33.3%) in the roxadustat and placebo arms, respectively, were TI responders (p = .217). A greater percentage of patients in the roxadustat arm with a transfusion burden of ≥2 pRBC units Q4W were TI responders (36.1%; 13/36) compared with the placebo arm (11.5%; 3/26; p-nominal = .047). The seven on-study deaths (4, roxadustat, and 3, placebo) were considered unrelated to treatment. Three roxadustat patients progressed to acute myeloid leukemia. Despite MATTERHORN not meeting its primary endpoint, a numerically higher TI rate was achieved with roxadustat treatment compared with placebo. Further analyses are needed to confirm the MDS patient subgroups deriving clinical benefit from this novel treatment.
  34. Cancer Lett. 2024 Jun 14. pii: S0304-3835(24)00454-3. [Epub ahead of print] 217060
      Leukemic stem cells (LSCs) in chronic myeloid leukemia (CML) contribute to treatment resistance and disease recurrence. Metabolism regulates LSCs, but the mechanisms remain elusive. Here, we show that hypoxia-inducible factor 2α (HIF-2α) is highly expressed in LSCs in mouse and human CML and increases after tyrosine kinase inhibitor (TKI) treatment. Deletion of HIF-2α suppresses disease progression, reduces LSC numbers, and enhances the efficacy of TKI treatment in BCL-ABL-induced CML mice. Mechanistically, HIF-2α deletion reshapes the metabolic profile of LSCs, leading to increased production of reactive oxygen species (ROS) and apoptosis in CML. Moreover, HIF-2α deletion decreases vascular endothelial growth factor (VEGF) expression, thereby suppressing neovascularization in the bone marrow of CML mice. Furthermore, pharmaceutical inhibition of HIF-2α by PT2399 attenuates disease progression and improves the efficacy of TKI treatment in both mouse and human CML. Overall, our findings highlight the role of HIF-2α in controlling the metabolic state and vascular niche remodeling in CML, suggesting it is a potential therapeutic target to enhance TKI therapy.
  35. Cell Rep. 2024 Jun 17. pii: S2211-1247(24)00706-X. [Epub ahead of print]43(7): 114378
      The Myb proto-oncogene encodes the transcription factor c-MYB, which is critical for hematopoiesis. Distant enhancers of Myb form a hub of interactions with the Myb promoter. We identified a long non-coding RNA (Myrlin) originating from the -81-kb murine Myb enhancer. Myrlin and Myb are coordinately regulated during erythroid differentiation. Myrlin TSS deletion using CRISPR-Cas9 reduced Myrlin and Myb expression and LDB1 complex occupancy at the Myb enhancers, compromising enhancer contacts and reducing RNA Pol II occupancy in the locus. In contrast, CRISPRi silencing of Myrlin left LDB1 and the Myb enhancer hub unperturbed, although Myrlin and Myb expressions were downregulated, decoupling transcription and chromatin looping. Myrlin interacts with the KMT2A/MLL1 complex. Myrlin CRISPRi compromised KMT2A occupancy in the Myb locus, decreasing CDK9 and RNA Pol II binding and resulting in Pol II pausing in the Myb first exon/intron. Thus, Myrlin directly participates in activating Myb transcription by recruiting KMT2A.
    Keywords:  CP: Molecular biology; H3K4me3; KMT2A; MLL1; Myb; Pol II pause release; enhancer RNA; enhancer hub; erythroid differentiation; gene expression; lncRNA
  36. Commun Biol. 2024 Jun 20. 7(1): 753
      Arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs) and is involved in various cellular processes, including cancer development. PRMT2 expression is increased in several cancer types although its role in acute myeloid leukemia (AML) remains unknown. Here, we investigate the role of PRMT2 in a cohort of patients with AML, PRMT2 knockout AML cell lines as well as a Prmt2 knockout mouse model. In patients, low PRMT2 expressors are enriched for inflammatory signatures, including the NF-κB pathway, and show inferior survival. In keeping with a role for PRMT2 in control of inflammatory signaling, bone marrow-derived macrophages from Prmt2 KO mice display increased pro-inflammatory cytokine signaling upon LPS treatment. In PRMT2-depleted AML cell lines, aberrant inflammatory signaling has been linked to overproduction of IL6, resulting from a deregulation of the NF-κB signaling pathway, therefore leading to hyperactivation of STAT3. Together, these findings identify PRMT2 as a key regulator of inflammation in AML.
  37. Sci Transl Med. 2024 Jun 19. 16(752): eadl5931
      Clinical treatment of acute myeloid leukemia (AML) largely relies on intensive chemotherapy. However, the application of chemotherapy is often hindered by cardiotoxicity. Patient sequence data revealed that angiotensin II receptor type 1 (AGTR1) is a shared target between AML and cardiovascular disease (CVD). We found that inhibiting AGTR1 sensitized AML to chemotherapy and protected the heart against chemotherapy-induced cardiotoxicity in a human AML cell-transplanted mouse model. These effects were regulated by the AGTR1-Notch1 axis in AML cells and cardiomyocytes from mice. In mouse cardiomyocytes, AGTR1 was hyperactivated by AML and chemotherapy. AML leukemogenesis increased the expression of the angiotensin-converting enzyme and led to increased production of angiotensin II, the ligand of AGTR1, in an MLL-AF9-driven AML mouse model. In this model, the AGTR1-Notch1 axis regulated a variety of genes involved with cell stemness and chemotherapy resistance. AML cell stemness was reduced after Agtr1a deletion in the mouse AML cell transplant model. Mechanistically, Agtr1a deletion decreased γ-secretase formation, which is required for transmembrane Notch1 cleavage and release of the Notch1 intracellular domain into the nucleus. Using multiomics, we identified AGTR1-Notch1 signaling downstream genes and found decreased binding between these gene sequences with Notch1 and chromatin enhancers, as well as increased binding with silencers. These findings describe an AML/CVD association that may be used to improve AML treatment.
  38. Clin Lymphoma Myeloma Leuk. 2024 May 15. pii: S2152-2650(24)00172-1. [Epub ahead of print]
      The 5th edition of the World Health Organization (WHO) classification of Hematolymphoid tumors provides a hierarchically-driven catalog of hematologic neoplasms and introduces a series of changes to the classification of acute myeloid leukemia (AML). Emphasizing molecular genetic findings, it expands the category of "acute myeloid leukemias with defining genetic abnormalities" while retaining the morphologically defined category of AML for cases that do not harbor disease-defining genetic drivers. The updates to the classification of AML provide refined definitions and diagnostic criteria based on clinicopathologic parameters and molecular genetic findings, emphasizing therapeutically and/or prognostically actionable biomarkers. This review provides an overview of the WHO 5th classification for AML with practical considerations for applying this classification system.
    Keywords:  AML; Genetic; Molecular; Prognosis
  39. Nat Commun. 2024 Jun 20. 15(1): 5272
      While myelodysplastic syndromes with del(5q) (del(5q) MDS) comprises a well-defined hematological subgroup, the molecular basis underlying its origin remains unknown. Using single cell RNA-seq (scRNA-seq) on CD34+ progenitors from del(5q) MDS patients, we have identified cells harboring the deletion, characterizing the transcriptional impact of this genetic insult on disease pathogenesis and treatment response. Interestingly, both del(5q) and non-del(5q) cells present similar transcriptional lesions, indicating that all cells, and not only those harboring the deletion, may contribute to aberrant hematopoietic differentiation. However, gene regulatory network (GRN) analyses reveal a group of regulons showing aberrant activity that could trigger altered hematopoiesis exclusively in del(5q) cells, pointing to a more prominent role of these cells in disease phenotype. In del(5q) MDS patients achieving hematological response upon lenalidomide treatment, the drug reverts several transcriptional alterations in both del(5q) and non-del(5q) cells, but other lesions remain, which may be responsible for potential future relapses. Moreover, lack of hematological response is associated with the inability of lenalidomide to reverse transcriptional alterations. Collectively, this study reveals transcriptional alterations that could contribute to the pathogenesis and treatment response of del(5q) MDS.
  40. Blood. 2024 Jun 20. pii: blood.2023023078. [Epub ahead of print]
      X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP) are uncommon diseases caused by loss-of-function and gain-of-function mutations, respectively, in the erythroid form of 5-aminolevulinic acid synthetase, ALAS2, which encodes the first enzyme in heme biosynthesis. A related sideroblastic anemia is due to mutations in SLC25A38, which supplies mitochondrial glycine for ALAS2 (SLC25A38-CSA). The lack of viable animal models has limited studies on the pathophysiology and development of therapies for these conditions. Here, using CRISPR-CAS9 gene editing technology, we have generated knock-in mouse models that recapitulate the main features of XLSA and XLPP, and, using conventional conditional gene targeting in embryonic stem cells, we also developed a faithful model of the SLC25A38-CSA. In addition to examining the phenotypes and natural history of each disease, we determine the effect of restriction or supplementation of dietary pyridoxine (vitamin B6), the essential cofactor of ALAS2, on the anemia and porphyria. In addition to the well-documented response of XLSA mutations to pyridoxine supplementation, we also demonstrate the relative insensitivity of the XLPP porphyria, severe sensitivity of the XLSA models, and an extreme hypersensitivity of the SLC25A38-CSA model to pyridoxine deficiency, a phenotype that is not shared with another mouse hereditary anemia model, Hbbth3/+ -thalassemia intermedia. Thus, in addition to generating animal models useful for examining the pathophysiology and treatment of these diseases, we have uncovered an unsuspected conditional synthetic lethality between the heme synthesis-related CSAs and pyridoxine deficiency. These findings have the potential to inform novel therapeutic paradigms for the treatment of these diseases.
  41. Nat Chem Biol. 2024 Jun 21.
      Metabolic alterations in cancer precipitate in associated dependencies that can be therapeutically exploited. To meet this goal, natural product-inspired small molecules can provide a resource of invaluable chemotypes. Here, we identify orpinolide, a synthetic withanolide analog with pronounced antileukemic properties, via orthogonal chemical screening. Through multiomics profiling and genome-scale CRISPR-Cas9 screens, we identify that orpinolide disrupts Golgi homeostasis via a mechanism that requires active phosphatidylinositol 4-phosphate signaling at the endoplasmic reticulum-Golgi membrane interface. Thermal proteome profiling and genetic validation studies reveal the oxysterol-binding protein OSBP as the direct and phenotypically relevant target of orpinolide. Collectively, these data reaffirm sterol transport as a therapeutically actionable dependency in leukemia and motivate ensuing translational investigation via the probe-like compound orpinolide.