bims-tremyl Biomed News
on Therapy resistance biology in myeloid leukemia
Issue of 2023–10–29
33 papers selected by
Paolo Gallipoli, Barts Cancer Institute, Queen Mary University of London



  1. Blood Adv. 2023 Oct 23. pii: bloodadvances.2023010563. [Epub ahead of print]
      Enasidenib is an inhibitor of isocitrate dehydrogenase 2 (IDH2) approved for the treatment of patients with IDH2-mutant relapsed/refractory acute myeloid leukemia (AML). In this phase 2/1b Beat AML sub-study, we applied a risk-adapted approach to assess the efficacy of enasidenib monotherapy for patients 60 years and older with newly diagnosed IDH2-mutant AML in whom genomic profiling demonstrated mutant IDH2 was in the dominant leukemic clone. Patients for whom enasidenib monotherapy did not induce a complete response (CR) or CR with incomplete blood count recovery (CRi) enrolled on a phase 1b cohort with the addition of azacitidine. The phase 2 portion assessing the overall response to enasidenib alone demonstrated efficacy, with a composite CR/CRi (cCR) rate of 46% in 60 evaluable patients. Seventeen patients subsequently transitioned to phase 1b combination therapy, with a cCR rate of 41% and one dose-limiting toxicity. Correlative studies highlight mechanisms of clonal elimination with differentiation therapy as well as therapeutic resistance. This study demonstrates both efficacy of enasidenib monotherapy in the upfront setting and feasibility and applicability of a risk-adapted approach to the upfront treatment of IDH2-mutant AML.
    DOI:  https://doi.org/10.1182/bloodadvances.2023010563
  2. Blood. 2023 Oct 27. pii: blood.2023021359. [Epub ahead of print]
      UBTF tandem duplications (UBTF-TDs) have recently emerged as a recurrent alteration in pediatric and adult acute myeloid leukemia (AML). UBTF-TD leukemias are characterized by a poor response to conventional chemotherapy and a transcriptional signature that mirrors NUP98-rearranged and NPM1-mutant AMLs, including HOX gene dysregulation. However, the mechanism of how UBTF-TD drives leukemogenesis remains unknown. In this study, we investigated the genomic occupancy of UBTF-TD in transformed cord-blood CD34+ (cbCD34+) cells and patient-derived xenograft models. We found that UBTF-TD protein maintained genomic occupancy at ribosomal DNA (rDNA) loci while also occupying genomic targets commonly dysregulated in UBTF-TD myeloid malignancies, such as the HOXA/HOXB gene clusters and MEIS1. These data suggest that UBTF-TD is a gain-of-function alteration that results in mislocalization to genomic loci dysregulated in UBTF-TD leukemias. UBTF-TD also co-occupies key genomic loci with KMT2A and Menin, which are known to be key partners involved in HOX-dysregulated leukemias. Using a protein degradation system, we showed that stemness, proliferation, and transcriptional signatures are dependent on sustained UBTF-TD localization to chromatin. Finally, we demonstrate that primary cells from UBTF-TD leukemias are sensitive to the Menin inhibitor SNDX-5613, resulting in markedly reduced in vitro and in vivo tumor growth, myeloid differentiation, and abrogation of the UBTF-TD leukemic expression signature. These findings provide a viable therapeutic strategy for patients with this high-risk AML subtype.
    DOI:  https://doi.org/10.1182/blood.2023021359
  3. Oncogene. 2023 Oct 23.
      Conventional therapies for acute myeloid leukemia (AML) often fail to eliminate the disease-initiating leukemia stem cell (LSC) population, leading to disease relapse. Interferon-γ (IFN-γ) is a known inflammatory cytokine that promotes antitumor responses. Here, we found that low serum IFN-γ levels correlated with a higher percentage of LSCs and greater relapse incidence in AML patients. Furthermore, IFNGR1 was overexpressed in relapsed patients with AML and associated with a poor prognosis. We showed that high doses (5-10 μg/day) of IFN-γ exerted an anti-AML effect, while low doses (0.01-0.05 μg/day) of IFN-γ accelerated AML development and supported LSC self-renewal in patient-derived AML-LSCs and in an LSC-enriched MLL-AF9-driven mouse model. Importantly, targeting the IFN-γ receptor IFNGR1 by using lentiviral shRNAs or neutralizing antibodies induced AML differentiation and delayed leukemogenesis in vitro and in mice. Overall, we uncovered essential roles for IFN-γ and IFNGR1 in AML stemness and showed that targeting IFNGR1 is a strategy to decrease stemness and increase differentiation in relapsed AML patients.
    DOI:  https://doi.org/10.1038/s41388-023-02874-5
  4. Mol Oncol. 2023 Oct 23.
      Leukaemia stem cells (LSCs) are the critical seed for the growth of haematological malignancies, driving the clonal expansion that enables disease initiation, relapse and often resistance. Specifically, they display inherent phenotypic and epigenetic plasticity resulting in complex heterogenic diseases. In this review, we discuss the key principles of deregulation of epigenetic processes that shape this disease evolution. We consider measures to define and quantify clonal heterogeneity, combining information from recent studies assessing mutational, transcriptional and epigenetic landscapes at single cell resolution in Myeloid Neoplasms (MN). We highlight the importance of integrating epigenetic and genetic information to better understand inter- and intra-patient heterogeneity and discuss how this understanding further informs evolution and progression trajectories and subsequent clinical response in MN. Under this topic, we also discuss efforts to identify mechanisms of resistance, by longitudinal analysis of patient samples. Finally, we highlight how we might target these aberrant epigenetic processes for better therapeutic outcomes and to potentially eradicate LSCs.
    Keywords:  Leukaemia stem cells; Myeloid neoplasms; epigenetic plasticity; heterogeneity; single cell studies and multiomics; targeting LSC
    DOI:  https://doi.org/10.1002/1878-0261.13544
  5. bioRxiv. 2023 Oct 10. pii: 2023.10.10.561642. [Epub ahead of print]
      Relapse of acute myeloid leukemia (AML) is highly aggressive and often treatment refractory. We analyzed previously published AML relapse cohorts and found that 40% of relapses occur without changes in driver mutations, suggesting that non-genetic mechanisms drive relapse in a large proportion of cases. We therefore characterized epigenetic patterns of AML relapse using 26 matched diagnosis-relapse samples with ATAC-seq. This analysis identified a relapse-specific chromatin accessibility signature for mutationally stable AML, suggesting that AML undergoes epigenetic evolution at relapse independent of mutational changes. Analysis of leukemia stem cell (LSC) chromatin changes at relapse indicated that this leukemic compartment underwent significantly less epigenetic evolution than non-LSCs, while epigenetic changes in non-LSCs reflected overall evolution of the bulk leukemia. Finally, we used single-cell ATAC-seq paired with mitochondrial sequencing (mtscATAC) to map clones from diagnosis into relapse along with their epigenetic features. We found that distinct mitochondrially-defined clones exhibit more similar chromatin accessibility at relapse relative to diagnosis, demonstrating convergent epigenetic evolution in relapsed AML. These results demonstrate that epigenetic evolution is a feature of relapsed AML and that convergent epigenetic evolution can occur following treatment with induction chemotherapy.
    DOI:  https://doi.org/10.1101/2023.10.10.561642
  6. bioRxiv. 2023 Oct 03. pii: 2023.10.02.560330. [Epub ahead of print]
      We previously reported that acute myeloid leukemia stem cells (LSCs) are uniquely reliant on oxidative phosphorylation (OXPHOS) for survival. Moreover, maintenance of OXPHOS is dependent on BCL2, creating a therapeutic opportunity to target LSCs using the BCL2 inhibitor drug venetoclax. While venetoclax-based regimens have indeed shown promising clinical activity, the emergence of drug resistance is prevalent. Thus, in the present study, we investigated how mitochondrial properties may influence mechanisms that dictate venetoclax responsiveness. Our data show that utilization of mitochondrial calcium is fundamentally different between drug responsive and non-responsive LSCs. By comparison, venetoclax-resistant LSCs demonstrate a more active metabolic (i.e., OXPHOS) status with relatively high steady-state levels of calcium. Consequently, we tested genetic and pharmacological approaches to target the mitochondrial calcium uniporter, MCU. We demonstrate that inhibition of calcium uptake sharply reduces OXPHOS and leads to eradication of venetoclax-resistant LSCs. These findings demonstrate a central role for calcium signaling in the biology of LSCs and provide a therapeutic avenue for clinical management of venetoclax resistance.
    Significance: We identify increased utilization of mitochondrial calcium as distinct metabolic requirement of venetoclax-resistant LSCs and demonstrate the potential of targeting mitochondrial calcium uptake as a therapeutic strategy.
    DOI:  https://doi.org/10.1101/2023.10.02.560330
  7. Nat Cancer. 2023 Oct 23.
      Despite recent advances in the treatment of acute myeloid leukemia (AML), there has been limited success in targeting surface antigens in AML, in part due to shared expression across malignant and normal cells. Here, high-density immunophenotyping of AML coupled with proteogenomics identified unique expression of a variety of antigens, including the RNA helicase U5 snRNP200, on the surface of AML cells but not on normal hematopoietic precursors and skewed Fc receptor distribution in the AML immune microenvironment. Cell membrane localization of U5 snRNP200 was linked to surface expression of the Fcγ receptor IIIA (FcγIIIA, also known as CD32A) and correlated with expression of interferon-regulated immune response genes. Anti-U5 snRNP200 antibodies engaging activating Fcγ receptors were efficacious across immunocompetent AML models and were augmented by combination with azacitidine. These data provide a roadmap of AML-associated antigens with Fc receptor distribution in AML and highlight the potential for targeting the AML cell surface using Fc-optimized therapeutics.
    DOI:  https://doi.org/10.1038/s43018-023-00656-2
  8. Blood. 2023 Oct 25. pii: blood.2023022330. [Epub ahead of print]
      The detection of measurable residual disease (MRD) is the strongest predictor of relapse in acute lymphoblastic leukemia (ALL). Using inotuzumab ozogamicin in the setting of MRD may improve outcomes. Patients with ALL in first complete remission (CR1) or beyond (CR2+) with MRD ≥1x10-4 were enrolled in this phase II trial. Inotuzumab was administered at 0.6 mg/m2 on Day 1 and 0.3 mg/m2 on Day 8 of cycle 1, then at 0.3 mg/m2 on Days 1 and 8 of cycles 2-6. Twenty-six consecutive patients with a median age of 46 years (range, 19-70) were treated. Nineteen (73%) were in CR1 and seven (27%) in CR2+; 16 (62%) had Philadelphia chromosome-positive ALL. Fifteen (58%) had baseline MRD ≥1x10-3. A median of three cycles (range, 1-6) were administered. Eighteen (69%) patients responded and achieved MRD negativity. After a median follow-up of 24 months (range, 9-43), the 2-year relapse-free survival (RFS) rate was 54% and the 2-year overall survival rate was 60% in the entire cohort. Most adverse events were low grade; sinusoidal obstruction syndrome was noted in two (8%) patients. In summary, inotuzumab ozogamicin resulted in favorable survival, MRD-negativity rates, and safety profile in patients with ALL and MRD-positive status. This study is registered at ClinicalTrials.gov (NCT03441061).
    DOI:  https://doi.org/10.1182/blood.2023022330
  9. Clin Lab Med. 2023 Dec;pii: S0272-2712(23)00071-9. [Epub ahead of print]43(4): 657-667
      Myelodysplastic syndromes (MDS) are a group of myeloid neoplasms characterized by clonal hematopoiesis and abnormal maturation of hematopoietic cells, resulting in cytopenias. The transformation of MDS to acute myeloid leukemia (AML) reflects a progressive increase in blasts due to impaired maturation of the malignant clone, and thus MDS and many AML subtypes form a biological continuum rather than representing two distinct diseases. Recent data suggest that, in addition to previously described translocations, NPM1 mutations and KMT2A rearrangements are also AML-defining genetic alterations that lead to rapid disease progression, even if they present initially with less than  20% blasts. While some adult patients <20% blasts can be treated effectively with intensive AML-type chemotherapy, in the future, treatment of individual patients in this MDS/AML group will likely be dictated by genetic, biological, and patient-related factors rather than an arbitrary blast percentage.
    Keywords:  Acute myeloid leukemia; Genomics; Myelodysplastic syndrome; Outcome; Progression
    DOI:  https://doi.org/10.1016/j.cll.2023.07.001
  10. Oncogene. 2023 Oct 27.
      KMT2A-rearranged (KMT2A-R) is an aggressive and chemo-refractory acute leukemia which mostly affects children. Transcriptomics-based characterization and chemical interrogation identified kinases as key drivers of survival and drug resistance in KMT2A-R leukemia. In contrast, the contribution and regulation of phosphatases is unknown. In this study we uncover the essential role and underlying mechanisms of SET, the endogenous inhibitor of Ser/Thr phosphatase PP2A, in KMT2A-R-leukemia. Investigation of SET expression in acute myeloid leukemia (AML) samples demonstrated that SET is overexpressed, and elevated expression of SET is correlated with poor prognosis and with the expression of MEIS and HOXA genes in AML patients. Silencing SET specifically abolished the clonogenic ability of KMT2A-R leukemic cells and the transcription of KMT2A targets genes HOXA9 and HOXA10. Subsequent mechanistic investigations showed that SET interacts with both KMT2A wild type and fusion proteins, and it is recruited to the HOXA10 promoter. Pharmacological inhibition of SET by FTY720 disrupted SET-PP2A interaction leading to cell cycle arrest and increased sensitivity to chemotherapy in KMT2A-R-leukemic models. Phospho-proteomic analyses revealed that FTY720 reduced the activity of kinases regulated by PP2A, including ERK1, GSK3β, AURB and PLK1 and led to suppression of MYC, supporting the hypothesis of a feedback loop among PP2A, AURB, PLK1, MYC, and SET. Our findings illustrate that SET is a novel player in KMT2A-R leukemia and they provide evidence that SET antagonism could serve as a novel strategy to treat this aggressive leukemia.
    DOI:  https://doi.org/10.1038/s41388-023-02840-1
  11. Blood Adv. 2023 10 23. pii: bloodadvances.2023011003. [Epub ahead of print]
      Germline genetic variants alter the coding and enhancer sequences of GATA2, which encodes a master regulator of hematopoiesis. The conserved murine Gata2 enhancer (+9.5) promotes hematopoietic stem cell (HSC) genesis during embryogenesis. Heterozygosity for a single-nucleotide Ets motif variant in the human enhancer creates a bone marrow failure and acute myeloid leukemia predisposition termed GATA2 deficiency syndrome. The homozygous murine variant attenuates chemotherapy- and transplantation-induced hematopoietic regeneration, hematopoietic stem and progenitor cell (HSPC) response to inflammation, and HSPC mobilization with the therapeutic mobilizer G-CSF. As a Gata2 +9.5 variant attenuated G-CSF-induced HSPC expansion and mobilization, and HSC transplantation therapies require efficacious mobilization, we tested whether variation impacts mechanistically distinct mobilizers or only those operating through select pathways. In addition to affecting G-CSF activity, Gata2 variation compromised IL-8/CXCR2- and VLA-4/VCAM1-induced mobilization. While the variation did not disrupt HSPC mobilization mediated by plerixafor, which functions through CXCR4/CXCL12, homozygous and heterozygous variation attenuated mobilization efficacy of the clinically used plerixafor/G-CSF combination. The influence of non-coding variation on HSPC mobilization efficacy and function is important clinically since comprehensive non-coding variation is not commonly analyzed in patients. Furthermore, our mobilization-defective system offers unique utility for elucidating fundamental HSPC mechanisms.
    DOI:  https://doi.org/10.1182/bloodadvances.2023011003
  12. Cancer Sci. 2023 Oct 26.
      Genetic mutations in the isocitrate dehydrogenase (IDH) gene that result in a pathological enzymatic activity to produce oncometabolite have been detected in acute myeloid leukemia (AML) patients. While specific inhibitors that target mutant IDH enzymes and normalize intracellular oncometabolite level have been developed, refractoriness and resistance has been reported. Since acquisition of pathological enzymatic activity is accompanied by the abrogation of the crucial WT IDH enzymatic activity in IDH mutant cells, aberrant metabolism in IDH mutant cells can potentially persist even after the normalization of intracellular oncometabolite level. Comparisons of isogenic AML cell lines with and without IDH2 gene mutations revealed two mutually exclusive signalings for growth advantage of IDH2 mutant cells, STAT phosphorylation associated with intracellular oncometabolite level and phospholipid metabolic adaptation. The latter came to light after the oncometabolite normalization and increased the resistance of IDH2 mutant cells to arachidonic acid-mediated apoptosis. The release of this metabolic adaptation by FDA-approved anti-inflammatory drugs targeting the metabolism of arachidonic acid could sensitize IDH2 mutant cells to apoptosis, resulting in their eradication in vitro and in vivo. Our findings will contribute to the development of alternative therapeutic options for IDH2 mutant AML patients who do not tolerate currently available therapies.
    Keywords:  acute myeloid leukemia; apoptosis; arachidonic acid; drug repositioning; phospholipid
    DOI:  https://doi.org/10.1111/cas.15994
  13. Crit Rev Oncol Hematol. 2023 Oct 23. pii: S1040-8428(23)00275-5. [Epub ahead of print] 104187
      Clonal hematopoiesis (CH) is defined by the presence of somatic mutations in hematopoietic stem and progenitor cells (HSPC). CH is associated primarily with advancing age and confers an elevated risk of progression to overt hematologic malignancy and cardiovascular disease. Increasingly, CH is associated with a wide range of diseases driven by, and sequelae of, inflammation. Accordingly, there is great interest in better understanding the pathophysiologic and clinical relationship between CH, aging, and disease. Both observational and experimental findings support the concept that CH is a potential common denominator in the inflammatory outcomes of aging. However, there is also evidence that local and systemic inflammatory states promote the growth and select for CH clones. In this review, we aim to provide an up-to-date summary of the nature of the relationship between inflammation and CH, which is central to unlocking potential therapeutic opportunities to prevent progression to myeloid malignancy.
    Keywords:  autoimmune disease; cardiovascular disease; clonal hematopoiesis; hematologic malignancy; inflammation; myeloid neoplasm
    DOI:  https://doi.org/10.1016/j.critrevonc.2023.104187
  14. Blood Adv. 2023 Oct 24. pii: bloodadvances.2023011389. [Epub ahead of print]
      Deleterious germline variants in DDX41 are a common cause of genetic predisposition to hematological malignancies, particularly myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML). Targeted next generation sequencing (NGS) was performed on large cohort of sequential patients with myeloid malignancy covering DDX41 as well as 30 other genes frequently mutated in myeloid malignancy. Whole genome transcriptome sequencing data was analyzed on a separate cohort of patients with a range of hematological malignancies to investigate the spectrum of cancer predisposition. 5737 patients with myeloid malignancies were studied with 152 different DDX41 variants detected. Multiple novel variants were detected, including synonymous variants affecting splicing as demonstrated by RNA-sequencing. The presence of a somatic DDX41 variant was highly associated with DDX41 germline variants in MDS and AML patients and we developed a statistical approach to incorporate the co-occurrence of a somatic DDX41 variant into germline variant classification at a "very strong" level (ACMG). Using this approach, the MDS cohort contained 3.8% (108/2865) patients with germline likely pathogenic/pathogenic (LP/P) variants and the AML cohort 4.9% (106/2157). DDX41 LP/P variants were markedly enriched in patients with AML and MDS compared to patients with myeloproliferative neoplasms (MPN), B-cell neoplasm and T- or B-cell acute lymphoblastic leukemia. In summary we have developed a framework to enhance DDX41 variant curation as well as highlighted the importance of assessment of all types of genomic variants (including synonymous and multi-exon deletions) in order to fully detect the landscape of possible clinically relevant DDX41 variants.
    DOI:  https://doi.org/10.1182/bloodadvances.2023011389
  15. Clin Lab Med. 2023 Dec;pii: S0272-2712(23)00074-4. [Epub ahead of print]43(4): 607-614
      The genetic underpinnings of myeloid neoplasms are becoming increasingly well understood. The accessibility to sequencing technology, in particular next-generation sequencing (NGS), has highlighted the importance of gene mutations in myelodysplastic syndromes (MDS) in conjunction with traditional cytogenetics. With the relatively recent influx of molecular information to complement known cytogenetic abnormalities, the diagnosis, classification, and prognosis of MDS and acute myeloid leukemia (AML) have been increasingly refined, which has also led to therapeutic advancements. It has been shown that TP53 mutations have a significant impact in cases of MDS, as well as AML, and have led to TP53-defined myeloid disease. TP53 mutations are also now incorporated into prognostic scoring systems, as patients have been shown to have aggressive disease and poor outcomes. With the increased understanding of the importance of TP53 disruption in myeloid neoplasia, it is likely that the critical role of TP53 will continue to be highlighted by an individual's disease classification and personalized therapeutic management.
    Keywords:  Cytogenetics; Mutations; Myelodysplasia; Myelodysplastic syndromes; Next generation sequencing; Prognosis; TP53
    DOI:  https://doi.org/10.1016/j.cll.2023.07.004
  16. Int J Mol Sci. 2023 Oct 22. pii: 15441. [Epub ahead of print]24(20):
      Acute myeloid leukemia (AML) with BCR::ABL1 has recently been recognized as a distinct subtype in international classifications. Distinguishing it from myeloid blast crisis chronic myeloid leukemia (BC-CML) without evidence of a chronic phase (CP), remains challenging. We aimed to better characterize this entity by integrating clonal architecture analysis, mutational landscape assessment, and gene expression profiling. We analyzed a large retrospective cohort study including CML and AML patients. Two AML patients harboring a BCR::ABL1 fusion were included in the study. We identified BCR::ABL1 fusion as a primary event in one patient and a secondary one in the other. AML-specific variants were identified in both. Real-time RT-PCR experiments demonstrated that CD25 mRNA is overexpressed in advanced-phase CML compared to AML. Unsupervised principal component analysis showed that AML harboring a BCR::ABL1 fusion was clustered within AML. An AML vs. myeloid BC-CML differential expression signature was highlighted, and while ID4 (inhibitor of DNA binding 4) mRNA appears undetectable in most myeloid BC-CML samples, low levels are detected in AML samples. Therefore, CD25 and ID4 mRNA expression might differentiate AML with BCR::ABL1 from BC-CML and assign it to the AML group. A method for identifying this new WHO entity is then proposed. Finally, the hypothesis of AML with BCR::ABL1 arising from driver mutations on a BCR::ABL1 background behaving as a clonal hematopoiesis mutation is discussed. Validation of our data in larger cohorts and basic research are needed to better understand the molecular and cellular aspects of AML with a BCR::ABL1 entity.
    Keywords:  AML; AML with BCR::ABL1; CD25; CML; ID4; RNA-Seq
    DOI:  https://doi.org/10.3390/ijms242015441
  17. J Clin Oncol. 2023 Oct 26. JCO2300546
       PURPOSE: Despite recent advances in adapting the intensity of treatment for older patients with ALL, current protocols are associated with high rates of early deaths, treatment-related toxicity, and dismal prognosis. We evaluated inotuzumab ozogamicin and dexamethasone (Dex) as induction therapy in older patients with ALL within the German Multicenter Study Group for Adult ALL (GMALL).
    PATIENTS AND METHODS: The open-label, multicenter, phase II, INITIAL-1 trial enrolled 45 patients older than 55 years with newly diagnosed, CD22-positive, BCR::ABL-negative B-precursor ALL (B-ALL). Patients received up to three cycles of inotuzumab ozogamicin/Dex and up to six cycles of age-adapted GMALL consolidation and maintenance therapy.
    RESULTS: Forty-three evaluable patients with common/pre-B (n = 38) and pro-B ALL (n = 5), with a median age of 64 years (range, 56-80), received at least two cycles of inotuzumab ozogamicin induction therapy. All patients achieved complete remission (CR/CR with incomplete hematologic recovery). Twenty-three (53%) and 30 (71%) patients had no evidence of molecularly assessed measurable residual disease (minimum 10e-4 threshold) after the second and third inductions, respectively. After a median follow-up of 2.7 years, event-free survival at one (primary end point) and 3 years was 88% (95% CI, 79 to 98) and 55% (95% CI, 40 to 71), while overall survival (OS) was 91% (95% CI, 82 to 99) and 73% (95% CI, 59 to 87), respectively. None of the patients died during 6 months after the start of induction. Most common adverse events having common toxicity criteria grade ≥3 during induction were leukocytopenia, neutropenia, thrombocytopenia, anemia, and elevated liver enzymes. One patient developed nonfatal veno-occlusive disease after induction II.
    CONCLUSION: Inotuzumab ozogamicin-based induction followed by age-adapted chemotherapy was well tolerated and resulted in high rates of remission and OS. These data provide a rationale for integrating inotuzumab ozogamicin into first-line regimens for older patients with B-ALL.
    DOI:  https://doi.org/10.1200/JCO.23.00546
  18. Blood Adv. 2023 Oct 23. pii: bloodadvances.2022009349. [Epub ahead of print]
      Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasm characterized by the accumulation of clonal mononuclear phagocyte system cells expressing CD1a and CD207. In the past decade, molecular profiling of LCH, as well as other histiocytic neoplasms demonstrated that these diseases are driven by MAP kinase (MAPK) activating alterations, with somatic BRAFV600E mutations in >50% of LCH patients, and clinical inhibition of MAPK signaling has demonstrated remarkable clinical efficacy. At the same time, activating alterations in kinase-encoding genes such as PIK3CA, ALK, RET, and CSF1R which can activate mitogenic pathways independent from the MAPK pathway have been reported in a subset of histiocytic neoplasms with anecdotal evidence of successful targeted treatment of histiocytoses harboring driver alterations in RET, ALK, and CSF1R. However, evidence supporting the biological consequences of expression of PIK3CA mutations in hematopoietic cells has been lacking, and whether targeted inhibition of PI3K is clinically efficacious in histiocytic neoplasms is unknown. Here, we provide evidence that activating mutations in PIK3CA can drive histiocytic neoplasms in vivo using a conditional knock-in mouse expressing mutant PIK3CAH1047R in monocyte/dendritic cell progenitors. In parallel, we demonstrate successful treatment of PIK3CA-mutated, multisystemic LCH using alpelisib, an inhibitor of the alpha catalytic subunit of PI3K. Alpelisib demonstrated a tolerable safety profile at a dose of 750mg/week and clinical and metabolic complete remission in a PIK3CA-mutated LCH patient. These data demonstrate PIK3CA as a targetable non-canonical driver of LCH and underscore the importance of mutational analysis-based personalized treatment in histiocytic neoplasms.
    DOI:  https://doi.org/10.1182/bloodadvances.2022009349
  19. BMC Cancer. 2023 Oct 26. 23(1): 1035
       BACKGROUND: Myelodysplastic Neoplasms (MDS) are clonal stem cell disorders characterized by ineffective hematopoiesis and progression to acute myeloid leukemia, myelodysplasia-related (AML-MR). A major mechanism of pathogenesis of MDS is the aberration of the epigenetic landscape of the hematopoietic stem cells and/or progenitor cells, especially DNA cytosine methylation, and demethylation. Data on TET2, the predominant DNA demethylator of the hematopoietic system, is limited, particularly in the MDS patients from India, whose biology may differ since these patients present at a relatively younger age. We studied the expression and the variants of TET2 in Indian MDS and AML-MR patients and their effects on 5-hydroxymethyl cytosine (5-hmC, a product of TET2 catalysis) and on the prognosis of MDS patients.
    RESULTS: Of the 42 MDS patients, cytogenetics was available for 31 sub-categorized according to the Revised International Prognostic Scoring System (IPSS-R). Their age resembled that of the previous studies from India. Bone marrow nucleated cells (BMNCs) were also obtained from 13 patients with AML-MR, 26 patients with de-novo AML, and 11 subjects with morphologically normal bone marrow. The patients had a significantly lower TET2 expression which was more pronounced in AML-MR and the IPSS-R higher-risk MDS categories. The 5-hmC levels in higher-risk MDS and AML-MR correlated with TET2 expression, suggesting a possible mechanistic role in the loss of TET2 expression. The findings on TET2 and 5-hmC were also confirmed at the tissue level using immunohistochemistry. Pathogenic variants of TET2 were found in 7 of 24 patient samples (29%), spanning across the IPSS-R prognostic categories. One of the variants - H1778R - was found to affect local and global TET2 structure when studied using structural predictions and molecular dynamics simulations. Thus, it is plausible that some pathogenic variants in TET2 can compromise the structure of TET2 and hence in the formation of 5-hmC.
    CONCLUSIONS: IPSS-R higher-risk MDS categories and AML-MR showed a reduction in TET2 expression, which was not apparent in lower-risk MDS. DNA 5-hmC levels followed a similar pattern. Overall, a decreased TET2 expression and a low DNA 5-hmC level are predictors of advanced disease and adverse outcome in MDS in the population studied, i.e., MDS patients from India.
    Keywords:  AML-myelodysplasia related; Acute myeloid leukemia with myelodysplasia related changes; DNA sequence analysis; Epigenetics; MDS in India; Molecular dynamics simulation; Myelodysplastic neoplasms; Myelodysplastic syndrome; TET2 protein
    DOI:  https://doi.org/10.1186/s12885-023-11449-2
  20. Nat Commun. 2023 Oct 23. 14(1): 6731
      Immunotherapies targeting cancer-specific neoantigens have revolutionized the treatment of cancer patients. Recent evidence suggests that epigenetic therapies synergize with immunotherapies, mediated by the de-repression of endogenous retroviral element (ERV)-encoded promoters, and the initiation of transcription. Here, we use deep RNA sequencing from cancer cell lines treated with DNA methyltransferase inhibitor (DNMTi) and/or Histone deacetylase inhibitor (HDACi), to assemble a de novo transcriptome and identify several thousand ERV-derived, treatment-induced novel polyadenylated transcripts (TINPATs). Using immunopeptidomics, we demonstrate the human leukocyte antigen (HLA) presentation of 45 spectra-validated treatment-induced neopeptides (t-neopeptides) arising from TINPATs. We illustrate the potential of the identified t-neopeptides to elicit a T-cell response to effectively target cancer cells. We further verify the presence of t-neopeptides in AML patient samples after in vivo treatment with the DNMT inhibitor Decitabine. Our findings highlight the potential of ERV-derived neoantigens in epigenetic and immune therapies.
    DOI:  https://doi.org/10.1038/s41467-023-42417-w
  21. Clin Epigenetics. 2023 Oct 26. 15(1): 171
       BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous disease with a poor prognosis. Dysregulation of the epigenetic machinery is a significant contributor to disease development. Some AML patients benefit from treatment with hypomethylating agents (HMAs), but no predictive biomarkers for therapy response exist. Here, we investigated whether unbiased genome-wide assessment of pre-treatment DNA-methylation profiles in AML bone marrow blasts can help to identify patients who will achieve a remission after an azacytidine-containing induction regimen.
    RESULTS: A total of n = 155 patients with newly diagnosed AML treated in the AMLSG 12-09 trial were randomly assigned to a screening and a refinement and validation cohort. The cohorts were divided according to azacytidine-containing induction regimens and response status. Methylation status was assessed for 664,227 500-bp-regions using methyl-CpG immunoprecipitation-seq, resulting in 1755 differentially methylated regions (DMRs). Top regions were distilled and included genes such as WNT10A and GATA3. 80% of regions identified as a hit were represented on HumanMethlyation 450k Bead Chips. Quantitative methylation analysis confirmed 90% of these regions (36 of 40 DMRs). A classifier was trained using penalized logistic regression and fivefold cross validation containing 17 CpGs. Validation based on mass spectra generated by MALDI-TOF failed (AUC 0.59). However, discriminative ability was maintained by adding neighboring CpGs. A recomposed classifier with 12 CpGs resulted in an AUC of 0.77. When evaluated in the non-azacytidine containing group, the AUC was 0.76.
    CONCLUSIONS: Our analysis evaluated the value of a whole genome methyl-CpG screening assay for the identification of informative methylation changes. We also compared the informative content and discriminatory power of regions and single CpGs for predicting response to therapy. The relevance of the identified DMRs is supported by their association with key regulatory processes of oncogenic transformation and support the idea of relevant DMRs being enriched at distinct loci rather than evenly distribution across the genome. Predictive response to therapy could be established but lacked specificity for treatment with azacytidine. Our results suggest that a predictive epigenotype carries its methylation information at a complex, genome-wide level, that is confined to regions, rather than to single CpGs. With increasing application of combinatorial regimens, response prediction may become even more complicated.
    Keywords:  AML; Azacytidine; DNA methylation patterns; DNA-methylation; Epigenetics; HMA-treatment; Predictive biomarker; Predictive signature
    DOI:  https://doi.org/10.1186/s13148-023-01580-z
  22. Sci Adv. 2023 Oct 27. 9(43): eadg5391
      Hematopoietic stem cells (HSCs) are tightly controlled to maintain a balance between blood cell production and self-renewal. While inflammation-related signaling is a critical regulator of HSC activity, the underlying mechanisms and the precise functions of specific factors under steady-state and stress conditions remain incompletely understood. We investigated the role of interferon regulatory factor 1 (IRF1), a transcription factor that is affected by multiple inflammatory stimuli, in HSC regulation. Our findings demonstrate that the loss of IRF1 from mouse HSCs significantly impairs self-renewal, increases stress-induced proliferation, and confers resistance to apoptosis. In addition, given the frequent abnormal expression of IRF1 in leukemia, we explored the potential of IRF1 expression level as a stratification marker for human acute myeloid leukemia. We show that IRF1-based stratification identifies distinct cancer-related signatures in patient subgroups. These findings establish IRF1 as a pivotal HSC controller and provide previously unknown insights into HSC regulation, with potential implications to IRF1 functions in the context of leukemia.
    DOI:  https://doi.org/10.1126/sciadv.adg5391
  23. Clin Cancer Res. 2023 Oct 27.
       PURPOSE: To assess the impact of PHF6 alterations on clinical outcome and therapeutical actionability in T cells acute lymphoblastic leukemia (T-ALL).
    EXPERIMENTAL DESIGN: We described PHF6 alterations in an adult cohort of T-ALL from the French trial GRAALL 2003/2005 and retrospectively analyzed clinical outcomes between PHF6-altered (PHF6ALT) and wild-type patients. We also used EPIC and ChIP-seq data of patient samples to analyze the epigenetic landscape of PHF6ALT T-ALLs. We consecutively evaluated 5-azacitidine efficacy, alone or combine with venetoclax, in PHF6ALT T-ALL.
    RESULTS: We show that PHF6 alterations account for 47% of cases in our cohort and demonstrate that PHF6ALT T-ALL presented significantly better clinical outcomes. Integrative analysis of DNA methylation and histone marks shows that PHF6ALT are characterized by DNA hypermethylation and H3K27me3 loss at promoters physiologically bivalent in thymocytes. Using patient-derived xenografts (PDX), we show that PHF6ALT T-ALL respond to the 5-azacytidine alone. Finally, synergism with the BCL2-inhibitor venetoclax was demonstrated in refractory/relapsing PHF6ALT T-ALL using fresh samples. Importantly, we report three cases of refractory/relapsed (R/R) PHF6ALT patients who were successfully treated with this combination.
    CONCLUSIONS: Overall, our study supports the use of PHF6 alterations as a biomarker of sensitivity to 5-azacytidine and venetoclax combination in R/R T-ALL.
    DOI:  https://doi.org/10.1158/1078-0432.CCR-23-2159
  24. Leuk Lymphoma. 2023 Oct 22. 1-9
      The independent prognostic significance of bone marrow fibrosis (BMF) in myelodysplastic syndromes (MDS) is challenged under currently molecular prognostic models. In this study, the clinical and genetic data from 438 MDS patients were analyzed retrospectively. The patients were randomly divided into training (n = 306) and validation (n = 132) cohorts. The independent significant prognostic factors included age, IPSS-R, BMF, TP53 and U2AF1. Using their weighted coefficients, we developed a simplified prognostic system. Four risk groups were produced: low, intermediate, high and very high. The new model yielded more clearly separated survival curves than the IPSS-R. In addition, our model achieved higher C-indexes (0.61 in the training cohort and 0.63 in the validation cohort) than the IPSS-RM model (0.59 and 0.58) and IPSS-R (0.57 and 0.56). In conclusion, BMF was an independent significant prognostic factor for MDS, and adding BMF into the IPSS-R improved its predictive capability.
    Keywords:  Myelodysplastic syndromes; TP53; bone marrow fibrosis; model; prognosis
    DOI:  https://doi.org/10.1080/10428194.2023.2271593
  25. Nucleic Acids Res. 2023 Oct 23. pii: gkad918. [Epub ahead of print]
      The BloodChIP Xtra database (http://bloodchipXtra.vafaeelab.com/) facilitates genome-wide exploration and visualization of transcription factor (TF) occupancy and chromatin configuration in rare primary human hematopoietic stem (HSC-MPP) and progenitor (CMP, GMP, MEP) cells and acute myeloid leukemia (AML) cell lines (KG-1, ME-1, Kasumi1, TSU-1621-MT), along with chromatin accessibility and gene expression data from these and primary patient AMLs. BloodChIP Xtra features significantly more datasets than our earlier database BloodChIP (two primary cell types and two cell lines). Improved methodologies for determining TF occupancy and chromatin accessibility have led to increased availability of data for rare primary cell types across the spectrum of healthy and AML hematopoiesis. However, there is a continuing need for these data to be integrated in an easily accessible manner for gene-based queries and use in downstream applications. Here, we provide a user-friendly database based around genome-wide binding profiles of key hematopoietic TFs and histone marks in healthy stem/progenitor cell types. These are compared with binding profiles and chromatin accessibility derived from primary and cell line AML and integrated with expression data from corresponding cell types. All queries can be exported to construct TF-gene and protein-protein networks and evaluate the association of genes with specific cellular processes.
    DOI:  https://doi.org/10.1093/nar/gkad918
  26. Nat Aging. 2023 Oct 26.
      DNA methylation deregulation at partially methylated domains (PMDs) represents an epigenetic signature of aging and cancer, yet the underlying molecular basis and resulting biological consequences remain unresolved. We report herein a mechanistic link between disrupted DNA methylation at PMDs and the spatial relocalization of H3K9me3-marked heterochromatin in aged hematopoietic stem and progenitor cells (HSPCs) or those with impaired DNA methylation. We uncover that TET2 modulates the spatial redistribution of H3K9me3-marked heterochromatin to mediate the upregulation of endogenous retroviruses (ERVs) and interferon-stimulated genes (ISGs), hence contributing to functional decline of aged HSPCs. TET2-deficient HSPCs retain perinuclear distribution of heterochromatin and exhibit age-related clonal expansion. Reverse transcriptase inhibitors suppress ERVs and ISGs expression, thereby restoring age-related defects in aged HSPCs. Collectively, our findings deepen the understanding of the functional interplay between DNA methylation and histone modifications, which is vital for maintaining heterochromatin function and safeguarding genome stability in stem cells.
    DOI:  https://doi.org/10.1038/s43587-023-00505-y
  27. Nat Commun. 2023 Oct 25. 14(1): 6402
      Lympho-hematopoiesis is regulated by cytokines; however, it remains unclear how cytokines regulate hematopoietic stem cells (HSCs) to induce production of lymphoid progenitors. Here, we show that in mice whose CXC chemokine ligand 12 (CXCL12) is deleted from half HSC niche cells, termed CXC chemokine ligand 12 (CXCL12)-abundant reticular (CAR) cells, HSCs migrate from CXCL12-deficient niches to CXCL12-intact niches. In mice whose CXCL12 is deleted from all Ebf3+/leptin receptor (LepR)+ CAR cells, HSCs are markedly reduced and their ability to generate B cell progenitors is reduced compared with that to generate myeloid progenitors even when transplanted into wild-type mice. Additionally, CXCL12 enables the maintenance of B lineage repopulating ability of HSCs in vitro. These results demonstrate that CAR cell-derived CXCL12 attracts HSCs to CAR cells within bone marrow and plays a critical role in the maintenance of HSCs, especially lymphoid-biased or balanced HSCs. This study suggests an additional mechanism by which cytokines act on HSCs to produce B cells.
    DOI:  https://doi.org/10.1038/s41467-023-42047-2
  28. Cell Stem Cell. 2023 Oct 16. pii: S1934-5909(23)00360-0. [Epub ahead of print]
      Hematopoietic stem cells (HSCs), which govern the production of all blood lineages, transition through a series of functional states characterized by expansion during fetal development, functional quiescence in adulthood, and decline upon aging. We describe central features of HSC regulation during ontogeny to contextualize how adaptive responses over the life of the organism ultimately form the basis for HSC functional degradation with age. We particularly focus on the role of cell cycle regulation, inflammatory response pathways, epigenetic changes, and metabolic regulation. We then explore how the knowledge of age-related changes in HSC regulation can inform strategies for the rejuvenation of old HSCs.
    Keywords:  aging; development; epigenetic; hematopoietic stem cells; inflammation; metabolism; niche regulation; quiescence; rejuvenation
    DOI:  https://doi.org/10.1016/j.stem.2023.09.013
  29. Blood. 2023 10 24. pii: blood.2023020079. [Epub ahead of print]
      The myelodysplastic syndromes (MDS) constitute a profoundly heterogeneous myeloid malignancy with a common origin in the hemopoietic stem cell compartment. Consequently, patient management and treatment are as heterogeneous. Decision-making includes identifying risk, symptoms, and options for the individual patient and to make a risk-benefit analysis. The only potential cure is allogeneic stem cell transplantation and albeit the fraction of transplanted MDS patients increase over time due to a better management and increased donor availability a majority are not eligible for this intervention. Current challenges encompass to decrease the relapse risk, the main cause of HSCT failure. Hypomethylating agents (HMA) constitute first-line treatment for higher-risk MDS. Combinations with other drugs as first-line treatment has to date not proven more efficacious than monotherapy, although combinations approved for acute myeloid leukemia, including venetoclax currently are under evaluation and often used as rescue treatment. The treatment goal for lower-risk MDS is to improve cytopenia, mainly anemia, quality-of-life, and possibly overall survival. Erythropoiesis-stimulating agents (ESAs) constitute first-line treatment for anemia and have better and more durable responses if initiated before the onset of a permanent transfusion need. Treatment in case of ESA failure or ineligibility should be tailored to the main disease mechanism; immunosuppression for hypoplastic MDS without high-risk genetics, lenalidomide for low-risk del(5q) MDS, and luspatercept for MDS with ring sideroblasts. Approved therapeutic options are still more scarce for MDS than for most other hematological malignancies. Better tools to match disease biology with treatment, i.e. applied precision medicine is needed to improve patient outcome.
    DOI:  https://doi.org/10.1182/blood.2023020079
  30. Blood. 2023 Oct 23. pii: blood.2023021126. [Epub ahead of print]
      Persisting alloreactive donor T cells in target tissues are a determinant of graft-versus-host disease (GVHD), but the transcriptional regulators that control the persistence and function of tissue-infiltrating T cells remain elusive. We demonstrate here that Id3, a DNA-binding inhibitor, is critical for sustaining T cell responses in GVHD target tissues in mice, including the liver and intestine. Id3 loss results in aberrantly expressed PD-1 in polyfunctional Th1 cells, decreased tissue-infiltrating PD-1+ polyfunctional Th1 cell numbers, impaired maintenance of liver TCF-1+ progenitor-like T cells, and inhibition of GVHD. PD-1 blockade restores the capacity of Id3-ablated donor T cells to mediate GVHD. Single-cell RNA-sequencing analysis revealed that Id3 loss leads to significantly decreased CD28- and PI3K/AKT-signaling activity in tissue-infiltrating polyfunctional Th1 cells, an indicator of active PD-1/PD-L1 effects. Id3 is also required for protecting CD8+ T cells from the PD-1 pathway-mediated suppression during GVHD. Genome-wide RNA-sequencing analysis reveals that Id3 represses transcription factors (e.g., Nfatc2, Fos, Jun, Ets1, Prdm1) that are critical for PD-1 transcription, exuberant effector differentiation, and IFN responses and dysfunction of activated T cells. Id3 achieves these effects by restraining the chromatin accessibility for these transcription factors. Id3 ablation in donor T cells preserved their graft-versus-tumor effects in mice undergoing allo-HSCT. Furthermore, CRIPSR/Cas9 knockout of ID3 in human CD19-directed CAR-T cells retained their anti-leukemia activity in NOD/SCID/IL2Rg-/- mice early after administration. These findings identify that ID3 is an important target to reduce GVHD, and gene-editing program of ID3 may have broad implications in T cell-based immunotherapy.
    DOI:  https://doi.org/10.1182/blood.2023021126
  31. bioRxiv. 2023 Oct 12. pii: 2023.10.11.561908. [Epub ahead of print]
      Chronic myeloid leukemia (CML) is initiated and initially maintained solely by the fusion gene BCR-ABL, encoding a multifaceted chimeric kinase targeted in the clinic with tyrosine kinase inhibitors (TKIs) TKIs are effective in inducing long-term remission, but are also frequently not curative. Thus, CML is an ideal system to test our hypothesis that transcriptome-based state-transition models accurately predict cancer evolution and treatment response. To test our hypothesis, we collected time-sequential blood samples from tetracycline-off (Tet-Off) BCR-ABL-inducible transgenic mice and wild-type controls. Using the time-series bulk RNA-seq analysis to capture a system-wide view of distinct disease states, we identified a single principal component that constructed a CML state-space with a three-well BCR-ABL leukemogenic potential landscape. The potential stable critical points defined observable disease states. Early states were characterized by anti-CML genes opposing leukemic transformation; late states were characterized by pro-CML genes. Genes with expression patterns shaped similarly to the potential landscape were identified as disease transition drivers. Re-introduction of tetracyclines to silence the BCR/ABL gene returned diseased mice transcriptomes to a stable state near to health, without reaching it, suggesting partly irreversible transformation changes. TKI treatment only reverted the diseased mice transcriptomes to an earlier disease state, without approaching health; disease relapse occurred soon after treatment completion. Using only the earliest time-point as initial conditions, our parametrized state-transition models accurately predicted both disease progression and treatment response, supporting this as a potentially valuable approach to time clinical intervention even before phenotypic changes become detectable.
    DOI:  https://doi.org/10.1101/2023.10.11.561908