bims-tremyl Biomed News
on Therapy resistance biology in myeloid leukemia
Issue of 2021‒09‒19
thirty papers selected by
Paolo Gallipoli
Barts Cancer Institute, Queen Mary University of London

  1. Mol Cancer Ther. 2021 Sep 13. pii: molcanther.0071.2021. [Epub ahead of print]
      Acute myeloid leukemia (AML) with a FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy associated with frequent relapse and poor overall survival. The tyrosine kinase inhibitor gilteritinib is approved for the treatment of relapse/refractory AML with FLT3 mutations, yet its mechanism of action is not completely understood. Here, we sought to identify additional therapeutic targets that can be exploited to enhance gilteritinib's anti-leukemic effect. Based on unbiased transcriptomic analyses, we identified the glutamine transporter SNAT1 (SLC38A1) as a novel target of gilteritinib that leads to impaired glutamine uptake and utilization within leukemic cells. Using metabolomics and metabolic flux analyses, we found that gilteritinib decreased glutamine metabolism through the TCA cycle and cellular levels of the oncometabolite 2-hydroxyglutarate. Additionally, gilteritinib treatment was associated with decreased ATP production and glutathione synthesis and increased reactive oxygen species, resulting in cellular senescence. Lastly, we found that the glutaminase inhibitor CB-839 enhanced anti-leukemic effect of gilteritinib in ex vivo studies using human primary FLT3-ITD-positive AML cells harboring mutations in the enzyme isocitrate dehydrogenase, which catalyzes the oxidative decarboxylation of isocitrate, producing α-ketoglutarate. Collectively, this work has identified a previously unrecognized, gilteritinib-sensitive metabolic pathway downstream of SLC38A1 that causes decreased glutaminolysis and disruption of redox homeostasis. These findings provide a rationale for the development and therapeutic exploration of targeted combinatorial treatment strategies for this subset of relapse/refractory AML.
  2. Cancer Discov. 2021 Sep 16. pii: candisc.1849.2020. [Epub ahead of print]
      An enhanced requirement for nutrients is a hallmark property of cancer cells. Here, we optimized an in vivo genetic screening strategy in acute myeloid leukemia (AML), which led to the identification of the myo-inositol transporter SLC5A3 as a dependency in this disease. We demonstrate that SLC5A3 is essential to support a myo-inositol auxotrophy in AML. The commonality among SLC5A3-dependent AML lines is the transcriptional silencing of ISYNA1, which encodes the rate limiting enzyme for myo-inositol biosynthesis, inositol-3-phosphate synthase 1. We use gain- and loss-of-function experiments to reveal a synthetic lethal genetic interaction between ISYNA1 and SLC5A3 in AML, which function redundantly to sustain intracellular myo-inositol. Transcriptional silencing and DNA hyper-methylation of ISYNA1 occur in a recurrent manner in human AML patient samples, in association with IDH1/IDH2 and CEBPA mutations. Our findings reveal myo-inositol as a nutrient dependency in AML caused by the aberrant silencing of a biosynthetic enzyme.
  3. Blood. 2021 Sep 15. pii: blood.2021013201. [Epub ahead of print]
      AML is characterized by the presence of leukemia stem cells (LSCs), and failure to fully eradicate this population contributes to disease persistence/relapse. Prior studies have characterized metabolic vulnerabilities of LSCs, which demonstrate preferential reliance on oxidative phosphorylation (OXPHOS) for energy metabolism and survival. In the present study, using both genetic and pharmacologic strategies in primary human AML specimens, we show that signal transducer and activator of transcription 3 (STAT3) mediates OXPHOS in LSCs. STAT3 regulates AML-specific expression of MYC, which in turn controls transcription of the neutral amino acid transporter SLC1A5. We show that genetic inhibition of MYC or SLC1A5 acts to phenocopy the impairment of OXPHOS observed with STAT3 inhibition, thereby establishing this axis as a regulatory mechanism linking STAT3 to energy metabolism. Inhibition of SLC1A5 reduces intracellular levels of glutamine, glutathione and multiple TCA metabolites, leading to reduced TCA cycle activity and inhibition of OXPHOS. Based on these findings, we used a novel small molecule STAT3 inhibitor, that binds STAT3 and disrupts STAT3-DNA, to evaluate the biological role of STAT3. We show that STAT3 inhibition selectively leads to cell death in AML stem and progenitor cells derived from newly diagnosed and relapsed patients, while sparing normal hematopoietic cells. Together, these findings establish a STAT3-mediated mechanism that controls energy metabolism and survival in primitive AML cells.
  4. Nat Commun. 2021 Sep 13. 12(1): 5397
      Acute myeloid leukemia (AML) remains incurable, largely due to its resistance to conventional treatments. Here, we find that increased abundance of the ubiquitin ligase RNF5 contributes to AML development and survival. High RNF5 expression in AML patient specimens correlates with poor prognosis. RNF5 inhibition decreases AML cell growth in culture, in patient-derived xenograft (PDX) samples and in vivo, and delays development of MLL-AF9-driven leukemogenesis in mice, prolonging their survival. RNF5 inhibition causes transcriptional changes that overlap with those seen upon histone deacetylase (HDAC)1 inhibition. RNF5 induces the formation of K29 ubiquitin chains on the histone-binding protein RBBP4, promoting its recruitment to and subsequent epigenetic regulation of genes involved in AML maintenance. Correspondingly, RNF5 or RBBP4 knockdown enhances AML cell sensitivity to HDAC inhibitors. Notably, low expression of both RNF5 and HDAC coincides with a favorable prognosis. Our studies identify an ERAD-independent role for RNF5, demonstrating that its control of RBBP4 constitutes an epigenetic pathway that drives AML, and highlight RNF5/RBBP4 as markers useful to stratify patients for treatment with HDAC inhibitors.
  5. Cancer Discov. 2021 Sep 16. pii: candisc.1851.2020. [Epub ahead of print]
      CRISPR-Cas9-based genetic screens have successfully identified cell type-dependent liabilities in cancer, including acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because most of these screens have been performed in vitro using established cell lines, evaluating the physiological relevance of these targets is critical. We have established a CRISPR screening approach using orthotopic xenograft models to validate and prioritize AML-enriched dependencies in vivo, including in CRISPR-competent AML patient-derived xenograft (PDX) models tractable for genome editing. Our integrated pipeline has revealed several targets with translational value, including SLC5A3 as a metabolic vulnerability for AML addicted to exogenous myo-inositol and MARCH5 as a critical guardian to prevent apoptosis in AML. MARCH5 repression enhanced the efficacy of BCL2 inhibitors such as venetoclax, further highlighting the clinical potential of targeting MARCH5 in AML. Our study provides a valuable strategy for discovery and prioritization of new candidate AML therapeutic targets.
  6. Blood Adv. 2021 Sep 17. pii: bloodadvances.2021004814. [Epub ahead of print]
      Prediction of resistant disease at initial diagnosis of acute myeloid leukemia (AML) can be achieved with high accuracy by using cytogenetic data and 29 gene expression markers (PS29MRC). Our aim was to establish PS29MRC as a clinically usable assay by using the widely implemented NanoString platform and further validate the classifier in a more recently treated patient cohort. 351 patients with newly diagnosed AML intensively treated within the AMLCG registry were analyzed. As a continuous variable, PS29MRC performed best in predicting induction failure in comparison to previously published risk models (OR=2.37; p=1.20·10-9). The classifier was strongly associated with overall survival (HR=1.38; p=2.62·10-6). We were able to establish a previously defined cut-off that allows a classifier dichotomization (PS29MRCdic). PS29MRCdic significantly identified induction failure with 59% sensitivity, 77% specificity and 72% overall accuracy (OR=4.81; p=4.15·10-10). PS29MRCdic was able to improve the ELN-2017 risk classification within every category (favorable: OR=5.44; p=0.017; intermediate: OR=4.43; p=0.011; adverse: OR=2.52; p=0.034). Median patients' overall survival with high PS29MRCdic was 1.8 years compared to 4.3 years of low-risk patients. In multivariate analysis including ELN-2017, clinical and genetic markers, only age and PS29MRCdic were independent predictors of refractory disease. In patients aged 60 or older, only PS29MRCdic was left as significant variable. In summary, we confirmed PS29MRC as a valuable classifier that can be calculated and reproduced on a widely available platform to identify high-risk patients in AML. Risk classification can still be refined beyond ELN-2017 and predictive classifiers might facilitate clinical trials focusing on these high-risk AML patients.
  7. Blood Adv. 2021 Sep 13. pii: bloodadvances.2020004136. [Epub ahead of print]
      The negative prognostic impact of FLT3-ITD in patients with acute myeloid leukemia with mutated NPM1 (AML-NPM1) is restricted to patients with a higher FLT3-ITD allelic ratio (≥0.5; FLT3high) and considered negligible in wild-type (FLT3wt)/low ITD ratio (FLT3low) patients. Since the co-mutation of DNMT3A (DNMT3Amut) has been suggested to negatively influence the prognosis of AML-NPM1, we analyzed DNMT3Amut impact in FLT3-ITD subsets (absent, low and high ratio). A total of 164 patients diagnosed with AML-NPM1 who received intensive chemotherapy according to two consecutive protocols (AML-03 and AML-12) were selected: 76 harboring FLT3-ITD (46%), 79 DNMT3Amut (48%), and 39 (24%) showing both mutations. Overall, DNMT3A mutational status did not show prognostic impact with comparable OS (mut vs. wt 62±6% vs. 56±6%; p=0.2), RR (22±11% vs. 31±11%; p=0.2) and LFS (65±6 vs. 54±6, p=0.1). Prognostic stratification established by FLT3-ITD (FLT3wt= FLT3low> FLT3high) was independent of DNMT3A mutational status. Measurable residual disease (MRD) based on NPM1 quantitative-PCR kinetics was available in 94 patients. DNMT3Amut was associated with a higher number of mutated NPM1 transcripts following induction (p=0.012) and first consolidation (C1; p<0.001). All DNMT3Amut patients were MRD positive following C1 (p<0.001) and exhibit significant MRD persistence after second and third consolidations (MRD positive vs. negative p=0.027 and p=0.001). Finally, DNMT3Amut patients presented a trend to greater risk of molecular relapse (p=0.054). When molecular failure was proven, patients underwent an allogeneic transplant. In conclusion, DNMT3Amut did not modify overall prognosis exerted by FLT3-ITD in AML-NPM1 despite delayed MRD clearance, possibly due to MRD-driven pre-emptive intervention.
  8. J Immunother Cancer. 2021 Sep;pii: e003149. [Epub ahead of print]9(9):
      BACKGROUND: Successful development of chimeric antigen receptor (CAR) T cell immunotherapy for children and adults with relapsed/refractory acute myeloid leukemia (AML) is highly desired given their poor clinical prognosis and frequent inability to achieve cure with conventional chemotherapy. Initial experiences with CD19 CAR T cell immunotherapy for patients with B-cell malignancies highlighted the critical impact of intracellular costimulatory domain selection (CD28 vs 4-1BB (CD137)) on CAR T cell expansion and in vivo persistence that may impact clinical outcomes. However, the impact of costimulatory domains on the efficacy of myeloid antigen-directed CAR T cell immunotherapy remains unknown.METHODS: In this preclinical study, we developed six CAR constructs targeting CD33, a highly expressed and validated AML target, comprised of one of three single-chain variable fragments with CD3ζ and either CD28 or 4-1BB costimulatory domains. We systematically compared the preclinical in vitro and in vivo efficacy of T cells lentivirally transduced with CD33 CAR constructs (CD33CARTs) against human AML.
    RESULTS: We observed potent in vitro cytokine production and cytotoxicity of CD33CARTs incubated with human CD33+ AML cell lines, as well as robust in vivo antileukemia activity in cell line and childhood AML patient-derived xenograft (PDX) models. Gemtuzumab-based CD33CARTs were unexpectedly toxic in vivo in animal models despite observed in vitro anti-leukemia activity. CD28-based CD33CARTs consistently induced more robust inhibition of leukemia proliferation in AML cell line and PDX models than did 4-1BB-based CD33CARTs. A 'best-in-class' lintuzumab-CD28/CD3ζ CAR construct was thus selected for clinical translation.
    CONCLUSIONS: CD33 is a critical antigen for potential immunotherapeutic targeting in patients with AML. Based on this rigorous preclinical evaluation, our validated clinical grade lintuzumab-CD28/CD3ζ CD33CART immunotherapy is now under evaluation in a first-in-child/first-in-human phase 1 clinical trial for children and adolescents/young adults with relapsed/refractory AML.
    Keywords:  adoptive; chimeric antigen; hematologic neoplasms; immunotherapy; pediatrics; receptors; translational medical research
  9. Blood Adv. 2021 Sep 17. pii: bloodadvances.2021004878. [Epub ahead of print]
      The contribution of the bone marrow (BM) immune microenvironment (TME) to acute myeloid leukemia (AML) development is well-known, but its prognostic significance is still elusive. Indoleamine 2,3-dioxygenase 1 (IDO1), which is negatively regulated by the BIN1 proto-oncogene, is an interferon (IFN)-γ-inducible mediator of immune tolerance. With the aim to develop a prognostic IDO1-based immune gene signature, biological and clinical data of 732 patients with newly diagnosed, non-promyelocytic AML were retrieved from public datasets and analyzed using established computational pipelines. Targeted transcriptomic profiles of 24 diagnostic BM samples were analyzed using the NanoString's nCounter platform. BIN1 and IDO1 were inversely correlated and individually predicted overall survival. PLXNC1, a semaphorin receptor involved in inflammation and immune response, was the IDO1-interacting gene retaining the strongest prognostic value. The incorporation of PLXNC1 into the 2-gene IDO1-BIN1 score gave rise to a powerful immune gene signature predicting survival, especially in patients receiving chemotherapy. The top differentially expressed genes between IDO1low and IDO-1high and between PLXNC1low and PLXNC1 high cases further improved the prognostic value of IDO1 providing a 7 and 10-gene immune signature, highly predictive of survival and correlating with AML mutational status at diagnosis. Taken together, our data indicate that IDO1 is pivotal for the construction of an immune gene signature predictive of survival in AML patients. Given the emerging role of immunotherapies for AML, our findings support the incorporation of immune biomarkers into current AML classification and prognostication algorithms.
  10. Cell Signal. 2021 Sep 09. pii: S0898-6568(21)00233-3. [Epub ahead of print]87 110144
      Cyclin A1 (CCNA1) is an alternative A-type cyclin that is expressed in acute myeloid leukemia (AML). However, its functions in AML cell chemoresistance, an important cause for mortality, are incompletely understood. The purpose of this study was to expound the role and potential mechanism of CCNA1 in AML cell chemoresistance. Upregulation of CCNA1 promoted resistance of AML cells to PKC412, AC220, and AraC. Mechanistically, it was confirmed that CCNA1 transcription was negatively regulated by forkhead box A2 (FOXA2), and the downregulation of FOXA2 promoted chemoresistance in AML cells. Moreover, the promoter sequence of CCNA1 has a significant H3K27me3 modification. Enhancer of zeste homolog 2 (EZH2) enhanced H3K27me3 modification of CCNA1 promoter to inhibit CCNA1 expression, thus promoting sensitivity of AML cells to drugs. Taken together, these findings lead to deeper insights into the mechanism of AML cell chemo-sensitivity by inhibiting CCNA1 at the transcriptional level.
    Keywords:  Acute myeloid leukemia; Cyclin A1; Enhancer of zeste homolog 2; FOXA2; H3K27me3
  11. Blood Adv. 2021 Sep 15. pii: bloodadvances.2021004435. [Epub ahead of print]
      The benefit of allogeneic hematopoietic stem cell transplantation (Allo-HSCT) for acute myeloid leukemia (AML) patients over 60 years remains a matter of debate, notably when performed in first complete remission (CR1). In order to clarify this issue, the French Innovative Leukemia Organization (FILO) performed a 10-year real-world time-dependent analysis. The study enrolled patients between 60 and 70 years of age with AML in CR1 after intensive chemotherapy with intermediate (IR) or unfavorable (UR) risk according to the European LeukemiaNet (ELN)-2010. The impact of Allo-HSCT was analyzed through three models, respectively i) time-dependent Cox, ii) multistate for dynamic prediction and iii) super landmark. The study enrolled 369 (73%) IR and 138 (27%) UR AML patients, 203 of whom received an Allo-HSCT. Classical multivariate analysis showed that Allo-HSCT significantly improved relapse-free (RFS; Hazard Ratio/HR [95%CI]: 0.47 [0.35-0.62], p<0.001) and overall (OS; HR [95%CI]: 0.56 [0.42-0.76], p<0.001) survivals, independently of the ELN risk group. With the multistate model, the predicted 5-year probability for IR and UR patients to remain in CR1 without Allo-HSCT was 8% and 1%, respectively. Dynamic predictions confirmed that patients without Allo-HSCT continue to relapse over time. Finally, the super landmark model showed that Allo-HSCT significantly improved RFS (HR [95%CI]: 0.47 [0.36-0.62], p<0.001) and OS (HR [95%CI]: 0.54 [0.40-0.72], p<0.001). Allo-HSCT in CR1 is demonstrated here to significantly improve the outcome of fit older AML patients. Long-term RFS without Allo-HSCT is very low (<10%), supporting Allo-HSCT as being the best curative option for these patients.
  12. Blood Cancer J. 2021 Sep 14. 11(9): 152
      Even though genetic perturbations and mutations are important for the development of myeloid malignancies, the effects of an inflammatory microenvironment are a critical modulator of carcinogenesis. Activation of the innate immune system through various ligands and signaling pathways is an important driver of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). The DAMPs, or alarmins, which activate the inflammasome pathway via the TLR4/NLR signaling cascade causes the lytic cell death of hematopoietic stem and progenitor cells (HSPCs), ineffective hematopoiesis, and β-catenin-induced proliferation of cancer cells, leading to the development of MDS/AML phenotype. It is also associated with other myeloid malignancies and involved in the pathogenesis of associated cytopenias. Ongoing research suggests the interplay of inflammasome mediators with immune modulators and transcription factors to have a significant role in the development of myeloid diseases, and possibly therapy resistance. This review discusses the role and importance of inflammasomes and immune pathways in myeloid malignancies, particularly MDS/AML, to better understand the disease pathophysiology and decipher the scope of therapeutic interventions.
  13. Blood Adv. 2021 Sep 15. pii: bloodadvances.2021004775. [Epub ahead of print]
      Patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) experience high rates of hospitalization, intensive care unit (ICU) admissions, and in-hospital deaths at end of life (EOL). Early goals-of-care (GOC) discussions might reduce intensity of care at EOL. Portable Medical Order (POLST) forms, known as Medical Orders for Life Sustaining Treatment (MOLST) forms in New York State, allow patients to translate GOC discussions into specific medical orders that communicate their wishes during a medical emergency. To determine if timing of MOLST form completion might be associated with EOL care in patients with AML and MDS. We conducted a retrospective study of 358 adult patients with AML and MDS treated at a single academic center and/or its affiliated sites and who died over a five year period. One-third of patients completed at least one MOLST form >30 days prior to death. Compared to patients who completed a MOLST form within 30 days of death or never completed a MOLST form, those who completed a MOLST form >30 days prior to death were less likely to receive transfusion [Adjusted Odds ratio (AOR) 0.39, p<0.01], chemotherapy (AOR 0.24, p<0.01), life-sustaining treatments (AOR 0.21, p<0.01), or to be admitted to the ICU (AOR 0.21, p<0.01) at EOL. They were also more likely to utilize hospice (AOR 2.72, p<0.01). Earlier MOLST form completion was associated with lower intensity of care at EOL in patients with MDS and AML.
  14. Blood Cancer Discov. 2021 Sep;2(5): 434-449
      Acute myeloid leukemia patients refractory to induction therapy or relapsed within one year have poor outcomes. Autocrine production of hepatocyte growth factor by myeloid blasts drives leukemogenesis in pre-clinical models. A phase Ib trial evaluated ficlatuzumab, a first-in-class anti-HGF antibody, in combination with cytarabine in this high-risk population. Dose-limiting toxicities were not observed, and 20 mg/kg was established as the recommended phase II dose. The most frequent treatment-related adverse event was febrile neutropenia. Among 17 evaluable patients, the overall response rate was 53%, all complete remissions. Phospho-proteomic mass cytometry showed potent on-target suppression of p-MET after ficlatuzumab treatment and that attenuation of p-S6 was associated with clinical response. Multiplexed single cell RNA sequencing using prospectively acquired patient specimens identified interferon response genes as adverse predictive factors. The ficlatuzumab and cytarabine combination is well-tolerated with favorable efficacy. High-dimensional analyses at single-cell resolution represent promising approaches for identifying biomarkers of response and mechanisms of resistance in prospective clinical studies.
    Keywords:  CyTOF; MET; ficlatuzumab; refractory AML; single cell RNA sequencing
  15. Blood Adv. 2021 Sep 14. pii: bloodadvances.2020003303. [Epub ahead of print]
      Although JAK1/2 inhibition is effective into alleviating symptoms of myelofibrosis (MF), it does not result in the eradication of MF clones, which can lead to inhibitor-resistant clones emerging during the treatment. Here we established iPS cells derived from MF patient samples (MF-iPSCs) harboring JAK2 V617F, CALR type 1, or CALR type 2 mutations. We demonstrated that these cells faithfully recapitulate the drug sensitivity of the disease. These cells were utilized for chemical screening and calcium/calmodulin-dependent protein kinase 2 (CAMK2) was identified as a promising therapeutic target. MF model cells and mice induced by MPL W515L, another type of mutations recurrently detected in MF patients were used to elucidate the therapeutic potential of CAMK2 inhibition. CAMK2 inhibition was effective against JAK2 inhibitor-sensitive and JAK2 inhibitor-resistant cells. Further research revealed CAMK2 gamma subtype was important in MF model cells induced by MPL W515L. We showed that CAMK2G hetero knockout in the primary bone marrow cells expressing MPL W515Ldecreased colony-forming capacity. CAMK2G inhibition with berbamine, a CAMK2G inhibitor, significantly prolonged survival and reduced disease phenotypes such as splenomegaly and leukocytosis in a MF mouse model induced by MPL W515L. We investigated the molecular mechanisms underlying the therapeutic effect of CAMK2G inhibition and found that CAMK2G is activated by MPL signaling in MF model cells and is an effector in the MPL-JAK2 signaling pathway in these cells. These results indicate CAMK2G plays an important role in MF, and CAMK2G inhibition may be a novel therapeutic strategy that overcomes resistance to JAK1/2 inhibition.
  16. Curr Opin Hematol. 2021 Sep 15.
      PURPOSE OF REVIEW: Relapsed disease is the primary cause of mortality for acute myeloid leukemia (AML) patients after allogeneic hematopoietic cell transplantation (HCT). This review outlines the most recent advances in the detection and prevention of AML relapse following allogeneic HCT.RECENT FINDINGS: Conventional methods for predicting post-HCT relapse rely on the molecular and cytogenetics features present at diagnosis. These methods are slow to reflect a growing understanding of the molecular heterogeneity of AML and impact of new therapies on post-HCT outcomes. The use of measurable residual disease (MRD) techniques, including multiparameter flow cytometry and molecular testing, may improve the prognostic ability of these models and should be incorporated into post-HCT surveillance whenever possible.In the post-HCT setting, FLT3 inhibitor maintenance data indicate that effective therapies can improve post-HCT outcomes. Maintenance data with DNA methyltransferase inhibitor monotherapy is less compelling and outcomes may improve with combinations. Early interventions directed at preemptive management of MRD may further improve post-HCT outcomes.
    SUMMARY: Post-HCT AML relapse prevention has evolved to include more sensitive measures of disease detection and novel therapies that may improve outcomes of poor-risk AML patients. Additional work is needed to maintain this progress.
  17. PLoS One. 2021 ;16(9): e0257090
      Isocitrate dehydrogenase 1 and 2 (IDH1/2) mutations and their key effector 2-hydroxyglutarate (2-HG) have been reported to promote oncogenesis in various human cancers. To elucidate molecular mechanism(s) associated with IDH1/2 mutations, we established mouse embryonic fibroblasts (MEF) cells and human colorectal cancer cells stably expressing cancer-associated IDH1R132C or IDH2R172S, and analyzed the change in metabolic characteristics of the these cells. We found that IDH1/2 mutants induced intracellular 2-HG accumulation and inhibited cell proliferation. Expression profile analysis by RNA-seq unveiled that glucose transporter 1 (Glut1) was induced by the IDH1/2 mutants or treatment with 2-HG in the MEF cells. Consistently, glucose uptake and lactate production were increased by the mutants, suggesting the deregulation of glucose metabolism. Furthermore, PI3K/Akt/mTOR pathway and Hif1α expression were involved in the up-regulation of Glut1. Together, these results suggest that Glut1 is a potential target regulated by cancer-associated IDH1/2 mutations.
  18. Blood Adv. 2021 Sep 17. pii: bloodadvances.2020004073. [Epub ahead of print]
      Donor lymphocyte infusion (DLI) is a standard of care for relapse of AML after allogeneic hematopoietic stem cell transplantation (aHSCT). Currently it is poorly understood how and when CD8+ αβ T cells exert graft-versus-leukemia (GvL) activity after DLI. Also, there is no reliable biomarker to monitor GvL activity of the infused CD8+ T cells. Therefore, we analyzed the dynamics of CD8+ αβ T cell clones in DLI-patients. In this prospective clinical study of 29 patients, we performed deep T cell receptor β (TRB) sequencing of sorted CD8+ αβ T cells to track patients' repertoire changes in response to DLI. Upon first occurrence of GvL, longitudinal analyses revealed a preferential expansion of distinct CD8+ TRB clones (n=14). This did not occur in samples of patients without signs of GvL (n=11). Importantly, early repertoire changes 15 days after DLI predicted durable remission for the 36 months study follow-up. Furthermore, absence of clonal outgrowth of the CD8+ TRB repertoire after DLI was an early biomarker that predicted relapse at a median time of 11.2 months ahead of actual diagnosis. Additionally, unbiased sample analysis regardless of the clinical outcome revealed that patients with decreasing CD8+ TRB diversity at day 15 after DLI (n=13) had a lower relapse incidence (P=0.0040) compared to patients without clonal expansion (n=6). In conclusion, CD8+ TRB analysis may provide a reliable tool for predicting the efficacy of DLI and holds the potential to identify patients at risk for progression and relapse after DLI.
  19. Nature. 2021 Sep 15.
      UTX (also known as KDM6A) encodes a histone H3K27 demethylase and is an important tumour suppressor that is frequently mutated in human cancers1. However, as the demethylase activity of UTX is often dispensable for mediating tumour suppression and developmental regulation2-8, the underlying molecular activity of UTX remains unknown. Here we show that phase separation of UTX underlies its chromatin-regulatory activity in tumour suppression. A core intrinsically disordered region (cIDR) of UTX forms phase-separated liquid condensates, and cIDR loss caused by the most frequent cancer mutation of UTX is mainly responsible for abolishing tumour suppression. Deletion, mutagenesis and replacement assays of the intrinsically disordered region demonstrate a critical role of UTX condensation in tumour suppression and embryonic stem cell differentiation. As shown by reconstitution in vitro and engineered systems in cells, UTX recruits the histone methyltransferase MLL4 (also known as KMT2D) to the same condensates and enriches the H3K4 methylation activity of MLL4. Moreover, UTX regulates genome-wide histone modifications and high-order chromatin interactions in a condensation-dependent manner. We also found that UTY, the Y chromosome homologue of UTX with weaker tumour-suppressive activity, forms condensates with reduced molecular dynamics. These studies demonstrate a crucial biological function of liquid condensates with proper material states in enabling the tumour-suppressive activity of a chromatin regulator.
  20. Nat Commun. 2021 Sep 17. 12(1): 5507
      The specific niche adaptations that facilitate primary disease and Acute Lymphoblastic Leukaemia (ALL) survival after induction chemotherapy remain unclear. Here, we show that Bone Marrow (BM) adipocytes dynamically evolve during ALL pathogenesis and therapy, transitioning from cellular depletion in the primary leukaemia niche to a fully reconstituted state upon remission induction. Functionally, adipocyte niches elicit a fate switch in ALL cells towards slow-proliferation and cellular quiescence, highlighting the critical contribution of the adipocyte dynamic to disease establishment and chemotherapy resistance. Mechanistically, adipocyte niche interaction targets posttranscriptional networks and suppresses protein biosynthesis in ALL cells. Treatment with general control nonderepressible 2 inhibitor (GCN2ib) alleviates adipocyte-mediated translational repression and rescues ALL cell quiescence thereby significantly reducing the cytoprotective effect of adipocytes against chemotherapy and other extrinsic stressors. These data establish how adipocyte driven restrictions of the ALL proteome benefit ALL tumours, preventing their elimination, and suggest ways to manipulate adipocyte-mediated ALL resistance.
  21. Blood. 2021 Sep 15. pii: blood.2021012130. [Epub ahead of print]
      Patients with severe aplastic anemia (SAA) are either treated with bone marrow transplant (BMT) or immunosuppression (IST) depending on their age, comorbidities, and available donors. In 2017, our phase 2 trial reported improved hematologic responses with the addition of eltrombopag (EPAG) to standard IST for SAA when compared to a historical cohort treated with IST alone. However, the rates and characteristics of long-term complications, relapse, and clonal evolution, previously described in patients treated with IST alone are not yet known with this new regimen, IST and EPAG. Patients were accrued from 2012 to 2020 with a total of 178 subjects included in this secondary endpoint analysis. With double the sample size and a much longer median follow-up (4 years) since the original report in 2017, we report a cumulative relapse rate of 39% in responding patients who received CSA maintenance, and clonal evolution of 15% in all treated patients at 4 years. Relapse occurred at distinct timepoints: after cyclosporine dose reduction and EPAG discontinuation at six months, and after two years when cyclosporine was discontinued. Most relapsed patients were retreated with therapeutic doses of cyclosporine +/- EPAG, and two-thirds responded. Clonal evolution to a myeloid malignancy or chromosome 7 abnormality (high-risk) was noted in 5.7% of patients and conferred a poorer overall survival. Neither relapse nor high-risk evolution occurred at a higher rate than was observed in a historical comparator cohort but the median time to both events were earlier in IST and EPAG treated patients. Trial registered in (NCT01623167).
  22. Blood Adv. 2021 Sep 16. pii: bloodadvances.2021005065. [Epub ahead of print]
      GATA2 Deficiency patients harbor de novo or inherited germline mutations in the GATA2 transcription factor gene, predisposing them to myeloid malignancies. There is considerable variation in disease progression, even among family members with the same mutation in GATA2. We investigated somatic mutations in 106 patients with GATA2 Deficiency to identify acquired mutations that are associated with myeloid malignancies. Myelodysplastic Syndrome (MDS) was the most common diagnosis (~44%), followed by GATA2 bone marrow immunodeficiency disorder (G2BMID) (~37%). Thirteen percent of the cohort had GATA2 mutations but displayed no disease manifestations. There were no correlations between patient age or sex with disease progression or survival. Cytogenetic analyses showed a high incidence of abnormalities (~43%)- notably trisomy 8 (~23%) and monosomy 7 (~12%), but these changes did not correlate with lower survival. Somatic mutations in ASXL1 and STAG2 were detected in ~25% of patients, though these mutations were rarely concomitant. Mutations in DNMT3A were found in ~10% of patients. These somatic mutations were found similarly in G2BMID and MDS, suggesting clonal hematopoiesis in early stages of disease, before the onset of MDS. ASXL1 mutations conferred a lower survival probability and were more prevalent in female patients. STAG2 mutations also conferred a lower survival probability, but did not show a statistically significant sex bias. There was a conspicuous absence of many commonly mutated genes associated with myeloid malignancies, including TET2, IDH1/2, and the splicing factor genes. Notably, somatic mutations in chromatin-related genes and cohesin genes characterized disease progression in GATA2 Deficiency.
  23. Mol Cancer Res. 2021 Sep 17. pii: molcanres.MCR-21-0456-E.2021. [Epub ahead of print]
      Exploitation of DNA repair defects has enabled major advances in treating specific cancers. Recent work discovered that the oncometabolite 2-hydroxyglutarate (2-HG), produced by neomorphic isocitrate dehydrogenase 1/2 (IDH1/2) mutations, confers a homology directed repair (HDR) defect through 2-HG-induced histone hypermethylation masking HDR signaling. Here, we report that IDH1 mutant cancer cells are profoundly sensitive to the histone deacetylase inhibitor (HDACi) vorinostat, by further suppressing the residual HDR in 2-HG-producing cells. Vorinostat down-regulates repair factors BRCA1 and RAD51 via disrupted E2F-factor regulation, causing increased DNA double strand breaks, reduced DNA repair factor foci, and functional HDR deficiency even beyond 2-HG's effects. This results in greater cell death of IDH1 mutant cells and confers synergy with radiation and PARPi, both against cells in culture and patient-derived tumor xenografts. Our work identifies HDACi's utility against IDH1 mutant cancers, and presents IDH1/2 mutations as potential biomarkers to guide trials testing HDACi in gliomas and other malignancies. Implications: IDH1 mutant cells show profound vulnerability to HDACi treatment, alone and with PARPi and radiation, via HDR suppression, presenting IDH1/2 mutations as biomarkers for HDACi's use in gliomas and other malignancies.
  24. Blood Adv. 2021 Sep 15. pii: bloodadvances.2021005117. [Epub ahead of print]
      Juvenile myelomonocytic leukemia is an overlapping myeloproliferative and myelodysplastic disorder of early childhood. It is associated with a spectrum of diverse outcomes ranging from spontaneous resolution in rare patients to transformation to acute myeloid leukemia in others that is generally fatal. This unpredictable clinical course, along with initially descriptive diagnostic criteria, led to decades of productive international research. Next generation sequencing now permits more accurate molecular diagnoses in nearly all patients. However, curative treatment is still reliant on allogeneic hematopoietic cell transplantation for most patients, and additional advances will be required to improve risk stratification algorithms that distinguish those that can be observed expectantly from others who require swift hematopoietic cell transplantation.
  25. Blood Adv. 2021 Sep 16. pii: bloodadvances.2021004638. [Epub ahead of print]
      Poly (ADP-ribose) polymerase-1 (PARP1) is a key mediator of various forms of DNA damage repair and plays an important role in the progression of several cancer types. The enzyme is activated by binding to DNA single-strand and double-strand breaks. Its contribution to chromatin remodeling makes PARP1 crucial for gene expression regulation. Inhibition of its activity with small molecules, leads to the synthetic lethal effect by impeding DNA repair in the treatment of cancer cells. At first PARP1 inhibitors (PARPi) were developed to target BRCA mutated cancer cells. Currently, PARPi are being studied to be used in a broader variety of patients either as single agents or in combination with chemotherapy, antiangiogenic agents, ionizing radiation, and immune checkpoint inhibitors. Ongoing clinical trials on olaparib, rucaparib, niraparib, veliparib and the most recent talazoparib show the advantage of these agents in overcoming PARPi resistance and underline their efficacy in targeted treatment of several hematologic malignancies. In this review, focusing on the crucial role of PARP1 in physiological and pathological effects in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), we give an outline of the enzyme's mechanisms of action and its role in the pathophysiology and prognosis of MDS/AML and we analyze the available data on the use of PARPi, highlighting their promising advances in clinical application.
  26. Cell Rep. 2021 Sep 14. pii: S2211-1247(21)01148-7. [Epub ahead of print]36(11): 109701
      Citrate lies at a critical node of metabolism, linking tricarboxylic acid metabolism and lipogenesis via acetyl-coenzyme A. Recent studies have observed that deficiency of the sodium-dependent citrate transporter (NaCT), encoded by SLC13A5, dysregulates hepatic metabolism and drives pediatric epilepsy. To examine how NaCT contributes to citrate metabolism in cells relevant to the pathophysiology of these diseases, we apply 13C isotope tracing to SLC13A5-deficient hepatocellular carcinoma (HCC) cells and primary rat cortical neurons. Exogenous citrate appreciably contributes to intermediary metabolism only under hypoxic conditions. In the absence of glutamine, citrate supplementation increases de novo lipogenesis and growth of HCC cells. Knockout of SLC13A5 in Huh7 cells compromises citrate uptake and catabolism. Citrate supplementation rescues Huh7 cell viability in response to glutamine deprivation or Zn2+ treatment, and NaCT deficiency mitigates these effects. Collectively, these findings demonstrate that NaCT-mediated citrate uptake is metabolically important under nutrient-limited conditions and may facilitate resistance to metal toxicity.
    Keywords:  NaCT; SLC13A5; citrate; hepatocellular carcinoma; lipogenesis; neurons; zinc