bims-tremyl Biomed News
on Therapy resistance biology in myeloid leukemia
Issue of 2022‒06‒12
29 papers selected by
Paolo Gallipoli
Barts Cancer Institute, Queen Mary University of London

  1. Nat Cell Biol. 2022 Jun 06.
      Mitochondrial metabolites regulate leukaemic and normal stem cells by affecting epigenetic marks. How mitochondrial enzymes localize to the nucleus to control stem cell function is less understood. We discovered that the mitochondrial metabolic enzyme hexokinase 2 (HK2) localizes to the nucleus in leukaemic and normal haematopoietic stem cells. Overexpression of nuclear HK2 increases leukaemic stem cell properties and decreases differentiation, whereas selective nuclear HK2 knockdown promotes differentiation and decreases stem cell function. Nuclear HK2 localization is phosphorylation-dependent, requires active import and export, and regulates differentiation independently of its enzymatic activity. HK2 interacts with nuclear proteins regulating chromatin openness, increasing chromatin accessibilities at leukaemic stem cell-positive signature and DNA-repair sites. Nuclear HK2 overexpression decreases double-strand breaks and confers chemoresistance, which may contribute to the mechanism by which leukaemic stem cells resist DNA-damaging agents. Thus, we describe a non-canonical mechanism by which mitochondrial enzymes influence stem cell function independently of their metabolic function.
  2. Biomark Res. 2022 Jun 10. 10(1): 43
      BACKGROUND: Immunotherapy of acute myeloid leukemia has experienced considerable advances, however novel target antigens continue to be sought after. To this end, unbiased approaches for surface protein detection are limited and integration with other data types, such as gene expression and somatic mutational burden, are poorly utilized. The Cell Surface Capture technology provides an unbiased, discovery-driven approach to map the surface proteins on cells of interest. Yet, direct utilization of primary patient samples has been limited by the considerable number of viable cells needed.METHODS: Here, we optimized the Cell Surface Capture protocol to enable direct interrogation of primary patient samples and applied our optimized protocol to a set of samples from patients with acute myeloid leukemia (AML) to generate the AML surfaceome. We then further curated this AML surfaceome to exclude antigens expressed on healthy tissues and integrated mutational burden data from hematologic cancers to further enrich for targets which are likely to be essential to leukemia biology. Finally, we validated our findings in a separate cohort of AML patient samples.
    RESULTS: Our protocol modifications allowed us to double the yield in identified proteins and increased the specificity from 54 to 80.4% compared to previous approaches. Using primary AML patient samples, we were able to identify a total of 621 surface proteins comprising the AML surfaceome. We integrated this data with gene expression and mutational burden data to curate a set of robust putative target antigens. Seventy-six proteins were selected as potential candidates for further investigation of which we validated the most promising novel candidate markers, and identified CD148, ITGA4 and Integrin beta-7 as promising targets in AML. Integrin beta-7 showed the most promising combination of expression in patient AML samples, and low or absent expression on healthy hematopoietic tissue.
    CONCLUSION: Taken together, we demonstrate the feasibility of a highly optimized surfaceome detection method to interrogate the entire AML surfaceome directly from primary patient samples and integrate this data with gene expression and mutational burden data to achieve a robust, multiomic target identification platform. This approach has the potential to accelerate the unbiased target identification for immunotherapy of AML.
    Keywords:  Acute myeloid leukemia; Immunology; Leukemia; Proteomics
  3. Leukemia. 2022 Jun 07.
      Acute myeloid leukemia (AML) is a heterogeneous group of aggressive hematological malignancies commonly associated with treatment resistance, high risk of relapse, and mitochondrial dysregulation. We identified six mitochondria-affecting compounds (PS compounds) that exhibit selective cytotoxicity against AML cells in vitro. Structure-activity relationship studies identified six analogs from two original scaffolds that had over an order of magnitude difference between LD50 in AML and healthy peripheral blood mononuclear cells. Mechanistically, all hit compounds reduced ATP and selectively impaired both basal and ATP-linked oxygen consumption in leukemic cells. Compounds derived from PS127 significantly upregulated production of reactive oxygen species (ROS) in AML cells and triggered ferroptotic, necroptotic, and/or apoptotic cell death in AML cell lines and refractory/relapsed AML primary samples. These compounds exhibited synergy with several anti-leukemia agents in AML, acute lymphoblastic leukemia (ALL), or chronic myelogenous leukemia (CML). Pilot in vivo efficacy studies indicate anti-leukemic efficacy in a MOLM14/GFP/LUC xenograft model, including extended survival in mice injected with leukemic cells pre-treated with PS127B or PS127E and in mice treated with PS127E at a dose of 5 mg/kg. These compounds are promising leads for development of future combinatorial therapeutic approaches for mitochondria-driven hematologic malignancies such as AML, ALL, and CML.
  4. Cancers (Basel). 2022 Jun 06. pii: 2817. [Epub ahead of print]14(11):
      This retrospective study investigated outcomes of 404 patients with relapsed/refractory (R/R) FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) acute myeloid leukemia (AML) enrolled in the PETHEMA registry, pre-approval of tyrosine kinase inhibitors. Most patients (63%) had received first-line intensive therapy with 3 + 7. Subsequently, patients received salvage with intensive therapy (n = 261), non-intensive therapy (n = 63) or supportive care only (n = 80). Active salvage therapy (i.e., intensive or non-intensive therapy) resulted in a complete remission (CR) or CR without hematological recovery (CRi) rate of 42%. More patients achieved a CR/CRi with intensive (48%) compared with non-intensive (19%) salvage therapy (p < 0.001). In the overall population, median overall survival (OS) was 5.5 months; 1- and 5-year OS rates were 25% and 7%. OS was significantly (p < 0.001) prolonged with intensive or non-intensive salvage therapy compared with supportive therapy, and in those achieving CR/CRi versus no responders. Of 280 evaluable patients, 61 (22%) had an allogeneic stem-cell transplant after they had achieved CR/CRi. In conclusion, in this large cohort study, salvage treatment approaches for patients with FLT3-ITD mutated R/R AML were heterogeneous. Median OS was poor with both non-intensive and intensive salvage therapy, with best long-term outcomes obtained in patients who achieved CR/CRi and subsequently underwent allogeneic stem-cell transplant.
    Keywords:  FLT3-ITD mutation; acute myeloid leukemia; real-world outcomes; relapsed/refractory disease; salvage therapy
  5. Blood. 2022 Jun 07. pii: blood.2021015135. [Epub ahead of print]
      Germline DDX41 variants are the most common mutations predisposing to acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) in adults, but the causal variant (CV) landscape and clinical spectrum of hematologic malignancies (HM) remain unexplored. Here, we analyzed the genomic profiles of 176 HM patients carrying 82 distinct presumably germline DDX41 variants among a group of 9,821 unrelated patients. Using our proposed DDX41 specific variant classification, we identified features distinguishing 116 HM patients with CV from 60 HM patients with variant of uncertain significance (VUS): an older age (median 69 years), male predominance (74% in CV versus 60% in VUS, P=0.03), frequent concurrent somatic DDX41 variants (79% in CV versus 5% in VUS, p<0.0001), a lower somatic mutation burden (1.4 ± 0.1 in CV versus 2.9 ± 0.04 in VUS, P=0.012), near exclusion of canonical recurrent genetic abnormalities including mutations in NPM1, CEBPA and FLT3 in AML, and favorable overall survival (OS) in AML/MDS patients. This superior OS was determined independent of blast count, abnormal karyotypes, and concurrent variants, including TP53 in AML/MDS patients, regardless of patient's sex, age or specific germline CV, suggesting that germline DDX41 variants define a distinct clinical entity. Furthermore, unrelated patients with myeloproliferative neoplasm (MPN) and B-cell lymphoma were linked by DDX41 CV, thus expanding the known disease spectrum. This study outlines the CV landscape, expands the phenotypic spectrum in unrelated DDX41-mutated patients, and underscores the urgent need for gene-specific diagnostic and clinical management guidelines.
  6. Cell Metab. 2022 Jun 07. pii: S1550-4131(22)00188-7. [Epub ahead of print]34(6): 801-802
      In this issue of Cell Metabolism, Liu et al. demonstrate that Prmt7 can regulate the onset and progression of leukemogenesis by inhibiting self-renewal capacity of leukemic stem cells (LSCs) as modeled in a murine version of chronic myeloid leukemia (CML).
  7. Blood. 2022 Jun 06. pii: blood.2021015173. [Epub ahead of print]
      To determine the survival benefit of allogeneic hematopoietic cell transplantation (allo-HCT) in chronic myelomonocytic leukemias (CMML), we assembled a retrospective cohort of CMML patients aged 18-70 years diagnosed between 2000 and 2014 from an International CMML Dataset (ICD, n=730) and from the EBMT registry (n=384). The prognostic impact of allo-HCT was analyzed through univariable and multivariable time-dependent models and with a multi-state model, accounting for age, sex, CMML prognostic scoring system (CPSS low and intermediate-1: lower-risk, intermediate-2 and high: higher-risk) at diagnosis, and AML transformation. In univariable analysis, lower-risk CMMLs had a 5-year OS of 20% (95%CI 12-33%) with allo-HCT versus 42% (95%CI 35-49%) without allo-HCT (P<0.001). In higher-risk patients, 5-year OS was 27% (95%CI 21-34%) with allo-HCT versus 15% (95%CI 11-22%) without allo-HCT (P=0.13). With multi-state models, performing allo-HCT before AML transformation reduced overall survival in patients with lower risk CMML while a survival benefit was predicted for men with higher risk CMML. In a multivariable analysis of lower-risk patients, performing allo-HCT before transformation to AML significantly increased the risk of death within two years of transplantation (HR=3.19, 95%CI 2.30-4.42, P<0.001), with no significant change in long-term survival beyond this time point (HR=0.98, 95%CI 0.58-1.64, P=0.92). In higher risk patients, allo-HCT significantly increased the risk of death in the first two years after transplant (HR=1.46, 95%CI 1.09-1.96, P=0.01), but not beyond (HR=0.60, 95%CI 0.34-1.08, P=0.09). Performing allo-HCT before AML transformation decreases life expectancy in lower risk patients but may be considered in higher risk patients.
  8. Haematologica. 2022 Jun 09.
      We retrospectively studied 125 AML patients with trisomy 4 (median age at diagnosis, 58 years; range, 16-77 years) treated between 2000 and 2019 within a multicenter study. Trisomy 4 was the sole abnormality in 28 (22%) patients and additional abnormalities were present in 97 (78%) patients. Twenty-two (22%) and 15 (15%) of 101 tested patients harbored NPM1 and FLT3-ITD mutations. Two (3%) of 72 tested patients were CEBPA double mutated. Data on response to intensive anthracycline-based induction therapy were available in 119 patients. Complete remission (CR) was achieved in 67% (n=80) and early death rate was 5% (n=6). Notably, patients with trisomy 4 as sole abnormality had a CR rate of 89%. An allo-HCT was performed in 40 (34%) patients, of whom 19 patients were transplanted in CR1. Median follow-up of the intensively treated cohort was 5.76 years (95%-CI, 2.99-7.61 years). Five-year overall survival (OS) and relapse-free survival were 30% (95%-CI, 22-41%) and 27% (95%-CI, 18-41%). An Andersen-Gill regression model on OS revealed ELN favorable-risk (HR, 0.34; P=0.006) and trisomy 4 as sole abnormality (HR, 0.41 P=0.01) as favorable factors, whereas age with a difference of ten years (HR, 1.15, P=0.11), female gender (OR, 0.74; P=0.20) and allo-HCT (OR, 0.64; P=0.14) had no significant impact. In our cohort, patients with trisomy 4 as a sole abnormality had a high CR rate and favorable clinical outcome. Allo-HCT seems not to improve OS.
  9. Blood Cancer J. 2022 Jun 09. 12(6): 91
      Recent advances in FLT3 and IDH targeted inhibition have improved response rates and overall survival in patients with mutations affecting these respective proteins. Despite this success, resistance mechanisms have arisen including mutations that disrupt inhibitor-target interaction, mutations impacting alternate pathways, and changes in the microenvironment. Here we review the role of these proteins in leukemogenesis, their respective inhibitors, mechanisms of resistance, and briefly ongoing studies aimed at overcoming resistance.
  10. Leukemia. 2022 Jun 10.
      Gemtuzumab ozogamicin (GO) is an anti-CD33 monoclonal antibody linked to calicheamicin, a DNA damaging agent, and is a well-established therapeutic for treating acute myeloid leukemia (AML). In this study, we used LASSO regression modeling to develop a 10-gene DNA damage response gene expression score (CalDDR-GEx10) predictive of clinical outcome in pediatric AML patients treated with treatment regimen containing GO from the AAML03P1 and AAML0531 trials (ADE + GO arm, N = 301). When treated with ADE + GO, patients with a high CalDDR-GEx10 score had lower complete remission rates (62.8% vs. 85.5%, P = 1.7 7 * 10-5) and worse event-free survival (28.7% vs. 56.5% P = 4.08 * 10-8) compared to those with a low CalDDR-GEx10 score. However, the CalDDR-GEx10 score was not associated with clinical outcome in patients treated with standard chemotherapy alone (ADE, N = 242), implying the specificity of the CalDDR-GEx10 score to calicheamicin-induced DNA damage response. In multivariable models adjusted for risk group, FLT3-status, white blood cell count, and age, the CalDDR-GEx10 score remained a significant predictor of outcome in patients treated with ADE + GO. Our findings present a potential tool that can specifically assess response to calicheamicin-induced DNA damage preemptively via assessing diagnostic leukemic cell gene expression and guide clinical decisions related to treatment using GO.
  11. Int J Mol Sci. 2022 May 25. pii: 5950. [Epub ahead of print]23(11):
      The European LeukemiaNet (ELN) criteria define the adverse genetic factors of acute myeloid leukemia (AML). AML with adverse genetic factors uniformly shows resistance to standard chemotherapy and is associated with poor prognosis. Here, we focus on the biological background and real-world etiology of these adverse genetic factors and then describe a strategy to overcome the clinical disadvantages in terms of targeting pivotal molecular mechanisms. Different adverse genetic factors often rely on common pathways. KMT2A rearrangement, DEK-NUP214 fusion, and NPM1 mutation are associated with the upregulation of HOX genes. The dominant tyrosine kinase activity of the mutant FLT3 or BCR-ABL1 fusion proteins is transduced by the AKT-mTOR, MAPK-ERK, and STAT5 pathways. Concurrent mutations of ASXL1 and RUNX1 are associated with activated AKT. Both TP53 mutation and mis-expressed MECOM are related to impaired apoptosis. Clinical data suggest that adverse genetic factors can be found in at least one in eight AML patients and appear to accumulate in relapsed/refractory cases. TP53 mutation is associated with particularly poor prognosis. Molecular-targeted therapies focusing on specific genomic abnormalities, such as FLT3, KMT2A, and TP53, have been developed and have demonstrated promising results.
    Keywords:  AML; ASXL1 mutation; BCR-ABL1 fusion; DEK-NUP214 fusion; ELN classification; FLT3-ITD with wild-type NPM1; KMT2A rearrangement; RUNX1 mutation; TP53 mutation; anti-CD47 antibody; complex karyotype; haploinsufficiency of GATA2 and mis-expression of MECOM; menin
  12. Nat Cancer. 2022 Jun 06.
      Selinexor is a first-in-class inhibitor of the nuclear exportin XPO1 that was recently approved by the US Food and Drug Administration for the treatment of multiple myeloma and diffuse large B-cell lymphoma. In relapsed/refractory acute myeloid leukemia (AML), selinexor has shown promising activity, suggesting that selinexor-based combination therapies may have clinical potential. Here, motivated by the hypothesis that selinexor's nuclear sequestration of diverse substrates imposes pleiotropic fitness effects on AML cells, we systematically catalog the pro- and anti-fitness consequences of selinexor treatment. We discover that selinexor activates PI3Kγ-dependent AKT signaling in AML by upregulating the purinergic receptor P2RY2. Inhibiting this axis potentiates the anti-leukemic effects of selinexor in AML cell lines, patient-derived primary cultures and multiple mouse models of AML. In a syngeneic, MLL-AF9-driven mouse model of AML, treatment with selinexor and ipatasertib outperforms both standard-of-care chemotherapy and chemotherapy with selinexor. Together, these findings establish drug-induced P2RY2-AKT signaling as an actionable consequence of XPO1 inhibition in AML.
  13. Mol Cancer. 2022 Jun 09. 21(1): 125
      BACKGROUND: The dynamic epigenome and proteins specialized in the interpretation of epigenetic marks critically contribute to leukemic pathogenesis but also offer alternative therapeutic avenues. Targeting newly discovered chromatin readers involved in leukemogenesis may thus provide new anticancer strategies. Accumulating evidence suggests that the PRC1 complex member CBX2 is overexpressed in solid tumors and promotes cancer cell survival. However, its role in leukemia is still unclear.METHODS: We exploited reverse genetic approaches to investigate the role of CBX2 in human leukemic cell lines and ex vivo samples. We also analyzed phenotypic effects following CBX2 silencing using cellular and molecular assays and related functional mechanisms by ATAC-seq and RNA-seq. We then performed bioinformatic analysis of ChIP-seq data to explore the influence of histone modifications in CBX2-mediated open chromatin sites. Lastly, we used molecular assays to determine the contribution of CBX2-regulated pathways to leukemic phenotype.
    RESULTS: We found CBX2 overexpressed in leukemia both in vitro and ex vivo samples compared to CD34+ cells. Decreased CBX2 RNA levels prompted a robust reduction in cell proliferation and induction of apoptosis. Similarly, sensitivity to CBX2 silencing was observed in primary acute myeloid leukemia samples. CBX2 suppression increased genome-wide chromatin accessibility followed by alteration of leukemic cell transcriptional programs, resulting in enrichment of cell death pathways and downregulation of survival genes. Intriguingly, CBX2 silencing induced epigenetic reprogramming at p38 MAPK-associated regulatory sites with consequent deregulation of gene expression.
    CONCLUSIONS: Our results identify CBX2 as a crucial player in leukemia progression and highlight a potential druggable CBX2-p38 MAPK network in AML.
    Keywords:  CBX2; Cancer; Chromatin readers; Epigenetics; Leukemia; PcG
  14. Front Oncol. 2022 ;12 863329
      Rearrangements of the Mixed Lineage Leukemia (MLL/KMT2A) gene are present in approximately 10% of acute leukemias and characteristically define disease with poor outcome. Driven by the unmet need to develop better therapies for KMT2A-rearranged leukemia, we previously discovered that the novel anti-cancer agent, curaxin CBL0137, induces decondensation of chromatin in cancer cells, delays leukemia progression and potentiates standard of care chemotherapies in preclinical KMT2A-rearranged leukemia models. Based on the promising potential of histone deacetylase (HDAC) inhibitors as targeted anti-cancer agents for KMT2A-rearranged leukemia and the fact that HDAC inhibitors also decondense chromatin via an alternate mechanism, we investigated whether CBL0137 could potentiate the efficacy of the HDAC inhibitor panobinostat in KMT2A-rearranged leukemia models. The combination of CBL0137 and panobinostat rapidly killed KMT2A-rearranged leukemia cells by apoptosis and significantly delayed leukemia progression and extended survival in an aggressive model of MLL-AF9 (KMT2A:MLLT3) driven murine acute myeloid leukemia. The drug combination also exerted a strong anti-leukemia response in a rapidly progressing xenograft model derived from an infant with KMT2A-rearranged acute lymphoblastic leukemia, significantly extending survival compared to either monotherapy. The therapeutic enhancement between CBL0137 and panobinostat in KMT2A-r leukemia cells does not appear to be mediated through cooperative effects of the drugs on KMT2A rearrangement-associated histone modifications. Our data has identified the CBL0137/panobinostat combination as a potential novel targeted therapeutic approach to improve outcome for KMT2A-rearranged leukemia.
    Keywords:  KMT2A-rearranged leukemia; chromatin; curaxin CBL0137; histone deacetylase inhibition; infant leukemia
  15. Leukemia. 2022 Jun 06.
      Pediatric acute myeloid leukemia (AML) develops from clonal expansion of hematopoietic precursor cells and is characterized by morphologic and cytomolecular heterogeneity. Although the past 40 years have seen significant improvements in overall survival, the prevailing treatment challenges in pediatric AML are the prevention of relapse and the management of relapsed disease. Approximately 25% of children and adolescents with AML suffer disease relapse and face a poor prognosis. Our greater understanding of the genomic, epigenomic, metabolomic, and immunologic pathophysiology of relapsed AML allows for better therapeutic strategies that are being developed for pediatric clinical trials. The development of biologically rational agents is critical as conventional chemotherapeutic salvage regimens are not effective for all patients and pose risk of organ toxicity in heavily pretreated patients. Another major barrier to improvement in outcomes for relapsed pediatric AML is the historic lack of availability and participation in clinical trials. There are ongoing efforts to launch multinational clinical trials of emerging therapies. The purpose of this review is to summarize currently available and newly developed therapies for relapsed pediatric AML.
  16. Adv Sci (Weinh). 2022 Jun;9(16): 2105811
      Mesenchymal stromal cells (MSCs) are essential elements of the bone marrow (BM) microenvironment, which have been widely implicated in pathways that contribute to leukemia growth and resistance. Recent reports showed genotypic and phenotypic alterations in leukemia patient-derived MSCs, indicating that MSCs might be educated/reprogrammed. However, the results have been inconclusive, possibly due to the heterogeneity of leukemia. Here, the authors report that acute myeloid leukemia (AML) induces MSCs towards an adipogenic differentiation propensity. RNAseq analysis reveal significant upregulation of gene expression enriched in the adipocyte differentiation process and reduction in osteoblast differentiation. The alteration is accompanied by a metabolic switch from glycolysis to a more oxidative phosphorylation-dependent manner. Mechanistic studies identify that AML cell-derived exosomes play a vital role during the AML cell-mediated MSCs education/reprogramming process. Pre-administration of mice BM microenvironment with AML-derived exosomes greatly enhance leukemia engraftment in vivo. The quantitative proteomic analysis identified a list of exosomal protein components that are differently expressed in AML-derived exosomes, which represent an opportunity for novel therapeutic strategies based on the targeting of exosome-based AML cells-MSCs communication. Collectively, the data show that AML-educated MSCs tend to differentiate into adipocytes contributing to disease progression, which suggests complex interactions of leukemia with microenvironment components.
    Keywords:  acute myeloid leukemia (AML); cell‐derived exosomes; mesenchymal stromal cells (MSCs)
  17. Clin Cancer Res. 2022 Jun 07. pii: clincanres.0809.2022-3-14 14:38:43.713. [Epub ahead of print]
      INTRODUCTION: We evaluated outcome of unrelated transplantation for primary refractory/relapsed (ref/rel) acute myeloid leukemia (AML) comparing two cohorts according to the year of transplant, 2000-2009 and 2010-2019.METHODS: Multivariable analyses were performed using the Cox proportional-hazards regression model.
    RESULTS: 3430 patients were included, 876 underwent a transplant between 2000-2009 and 2554 in 2010-2019. Median follow up was 8.7 (95% CI: 7.8-9.4) and 3.4 (95% CI: 3.1-3.6) years (p<0.001). Median age was 52 (18-77) and 56 (18-79) years (p<0.0001). 45.5% and 55.5% had refractory AML while 54.5% and 44.5 % had relapsed AML. Conditioning was myeloablative in 60% and 52%, respectively. Neutrophil recovery, day 100 incidence of acute and 2-year incidence of chronic graft-versus-host disease (GVHD) were similar between the two periods. Two-year relapse incidence was higher for patients transplanted in the 2000-2009 period vs. those transplanted in 2010-2019; 50.2% vs. 45.1%; (hazard ratio (HR)=0.85 (95% CI: 0.74-0.97), p=0. 002). Leukemia-free survival, overall survival and GVHD-free, relapse-free survival were lower for the 2000-2009 period, 26% vs. 32.1% (HR=0.87 (95% CI: 0.78-0.97), p=0.01), 32.1% vs. 38.1% (HR=0.86 (95% CI: 0.77-0.96), p=0.01) and 21.5% vs. 25.3% (HR=0.89 (95% CI: 0.81-0.99), p=0.03, respectively. Two-year non-relapse mortality was not significantly different, 23.8% vs. 23.7% (HR=0.91 (95% CI: 0.76-1.11), p=0.34.
    CONCLUSION: Outcome of unrelated transplantation for patients with ref/rel AML has improved in the last two decades, rescuing about one third of the patients.
  18. J Immunother Precis Oncol. 2021 Aug;4(3): 129-141
      Myelofibrosis (MF) is a myeloproliferative neoplasm hallmarked by uncontrolled blood counts, constitutional symptoms, extramedullary hematopoiesis, and an increased risk of developing acute myeloid leukemia. Janus kinase (JAK) inhibitors are the most common treatment for MF due to their ability to reduce spleen size and improve disease-related symptoms; however, JAK inhibitors are not suitable for every patient and their impact on MF is limited in several respects. Novel JAK inhibitors and JAK inhibitor combinations are emerging that aim to enhance the treatment landscape, providing deeper responses to a broader population of patients with the continued hope of providing disease modification and improving long-term outcomes. In this review, we highlight several specific areas of unmet need within MF. Subsequently, we review agents that target those areas of unmet need, focusing specifically on the JAK inhibitors, momelotinib, pacritinib, itacitinib, and NS-018 as well as JAK inhibitor combination approaches using CPI-0610, navitoclax, parsaclisib, and luspatercept.
    Keywords:  JAK inhibitor; myelofibrosis; myeloproliferative neoplasm; rare disease
  19. Blood Adv. 2022 Jun 08. pii: bloodadvances.2022007764. [Epub ahead of print]
      Promising results have been shown combining ponatinib and chemotherapy in adults with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL). The PONALFIL trial combined ponatinib (30 mg/day) with standard induction and consolidation chemotherapy followed by allogeneic hematopoietic stem cell transplant (alloHSCT) in newly diagnosed Ph+ALL patients aged 18-60 years. Ponatinib was only given pre-emptively after alloHSCT. Primary endpoints were hematologic and molecular response before alloHSCT and event-free survival (EFS), including molecular relapse as event. Thirty patients (median age 49 [19-59] years) entered the trial. All showed hematologic response, and alloHSCT was performed in 26 patients (20 in complete molecular response and 6 in major molecular response). Only one patient died by graft-versus-host disease and 5 patients showed molecular relapse after alloHSCT. No tyrosine kinase inhibitor (TKI) was given after HSCT in 18/26 patients. Twenty-nine patients are alive (median follow-up 2.1 years, range 0.2-4.0), with 3-year EFS and overall survival (OS) of 70% (95% CI, 51%-89%) and 96% (89%-100%). Comparison of the PONALFIL and the ALLPh08 trials (same schedule using imatinib as TKI ) by propensity score showed significant improvement in OS for patients in PONALFIL (3-year OS 96% vs. 53%, p=0.002). The most frequent grade 3-4 adverse events were hematologic (42%), infections (17%) and hepatic (22%), with only one vascular occlusive event. The combination of chemotherapy with ponatinib followed by alloHSCT is well tolerated, with encouraging EFS in adults with newly diagnosed Ph+ALL. Cross-trial comparison suggests improvement versus imatinib. ( NCT02776605).
  20. Blood Adv. 2022 Jan 06. pii: bloodadvances.2021006920. [Epub ahead of print]
      Chromosomal aberrations and gene mutations have been considered to be the major reasons for high recurrence rates and poor survival among AML patients. However, the underlying molecular mechanism of AML gene mutation remains largely unclear. Here, we show that sperm-associated antigen 6 (SPAG6), one of the most markedly increased SPAG genes in AML, significantly contributed to the proliferation and migration of leukemic cells. SPAG6 was highly expressed in AML and its upregulation was negatively correlated with the prognosis of the disease. In vitro, SPAG6 promoted the proliferation and migration of leukemia cells and promoted cell cycle progression from G1 phase to S phase. In vivo, low expression of SPAG6 reduced the proliferation and infiltration of leukemia cells and prolonged the survival of xenograft tumor mice. Furthermore, immunoprecipitation and mass spectrometry analysis showed that SPAG6 interacts with MYO1D. Specifically, overexpression of SPAG6 promoted the translocation of MYO1D into the cell membrane, thus upgrading the expression level of the EGFR family and thereby promoting the progression of AML. Overall, our study found that SPAG6 combined with MYO1D and translocated MYO1D from the cytosol to the cytomembrane, which induced the PI3K/AKT signaling and ERK signaling pathway to regulate the growth and prognosis of AML. SPAG6 may become a new target gene for the treatment of AML.
  21. Nat Commun. 2022 Jun 06. 13(1): 3131
      Human pluripotent stem cell differentiation towards hematopoietic progenitor cell can serve as an in vitro model for human embryonic hematopoiesis, but the dynamic change of epigenome and transcriptome remains elusive. Here, we systematically profile the chromatin accessibility, H3K4me3 and H3K27me3 modifications, and the transcriptome of intermediate progenitors during hematopoietic progenitor cell differentiation in vitro. The integrative analyses reveal sequential opening-up of regions for the binding of hematopoietic transcription factors and stepwise epigenetic reprogramming of bivalent genes. Single-cell analysis of cells undergoing the endothelial-to-hematopoietic transition and comparison with in vivo hemogenic endothelial cells reveal important features of in vitro and in vivo hematopoiesis. We find that JUNB is an essential regulator for hemogenic endothelium specialization and endothelial-to-hematopoietic transition. These studies depict an epigenomic roadmap from human pluripotent stem cells to hematopoietic progenitor cells, which may pave the way to generate hematopoietic progenitor cells with improved developmental potentials.
  22. Blood. 2022 Jun 08. pii: blood.2022016084. [Epub ahead of print]
      Hematopoietic stem/progenitor cells (HSPCs) reside in localized microenvironments, or niches, in the bone marrow that provide key signals regulating their activity. A fundamental property of hematopoiesis is the ability to respond to environmental cues, such as inflammation. How these cues are transmitted to HSPCs within hematopoietic niches is not well established. Here, we show that perivascular bone marrow dendritic cells (DCs) express a high basal level of toll-like receptor-1 (TLR1) and TLR2. Systemic treatment with a TLR1/2 agonist induces HSPC expansion and mobilization. It also induces marked alterations in the bone marrow microenvironment, including a decrease in osteoblast activity and sinusoidal endothelial cell number. TLR1/2 agonist treatment of mice in which Myd88 is deleted specifically in DCs using Zbtb46-Cre show that the TLR1/2-induced expansion of multipotent HPSCs, but not HSPC mobilization or alterations in the bone marrow microenvironment, are dependent on TLR1/2 signaling in DCs. Interleukin-1 beta (IL-1b) is constitutively expressed in both murine and human DCs and is further induced after TLR1/2 stimulation. Systemic TLR1/2 agonist treatment of Il1r1-/- mice show that TLR1/2-induced HSPC expansion is dependent on IL-1b signaling. Single cell RNA sequencing of low risk MDS bone marrow show that IL1B and TLR1 expression is increased in DCs. Collectively, these data suggest a model in which TLR1/2 stimulation of DCs induces secretion of IL-1b and other inflammatory cytokines into the perivascular niche, which in turn, regulates multipotent HSPCs. Increased DC TLR1/2 signaling may contribute to altered HSPC function in MDS by increasing local IL-1b expression.
  23. Leukemia. 2022 Jun 03.
      Eligibility criteria for clinical trials are intended to select suitable study subjects but can limit trial participation and generalization of results. While reported for other cancers, non-enrollment rates and evolution of eligibility criteria over time have so far not been studied for randomized controlled trials (RCTs) involving adults with acute myeloid leukemia (AML). Among 3698 studies published between 2010 and 2020, including 447 involving prospective clinical trials, we identified 75 phase three RCTs testing non-transplant therapies for adults with AML. Only 31 studies (41%) provided information on non-enrollment; in these studies, the median non-enrollment rate was 23%, primarily attributed to restrictive eligibility criteria. In 95% of trials, eligibility criteria were reported with the total number per trial increasing over time (P < 0.001), particularly in industry-funded trials. A total of 27 eligibility criteria were used across trials, mostly concerning comorbidities or performance status, with eight of them becoming more common over time. The concordance with recent ASCO - Friends of Cancer Research eligibility criteria recommendations greatly varied, from 35% to 99%. Together, our analyses suggest that the ability to generalize results from non-transplant RCTs may be increasingly limited because of high non-enrollment rates and increasingly restrictive eligibility criteria.
  24. Cell Stem Cell. 2022 Jun 02. pii: S1934-5909(22)00206-5. [Epub ahead of print]29(6): 882-904
      Clonal hematopoiesis of indeterminate potential (CHIP) describes a widespread expansion of genetically variant hematopoietic cells that increases exponentially with age and is associated with increased risks of cancers, cardiovascular disease, and other maladies. Here, we discuss how environmental contexts associated with CHIP, such as old age, infections, chemotherapy, or cigarette smoking, alter tissue microenvironments to facilitate the selection and expansion of specific CHIP mutant clones. Further, we consider major remaining gaps in knowledge, including intrinsic effects, clone size thresholds, and factors affecting clonal competition, that will determine future application of this field in transplant and preventive medicine.
    Keywords:  aging; cancer; chemotherapy; clonal competition; clonal hematopoiesis; infection; inflammation; radiation
  25. Cell. 2022 Jun 02. pii: S0092-8674(22)00597-9. [Epub ahead of print]
      A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (CRISPR-based screens with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, to date, have been used at limited scales. Here, we perform genome-scale Perturb-seq targeting all expressed genes with CRISPR interference (CRISPRi) across >2.5 million human cells. We use transcriptional phenotypes to predict the function of poorly characterized genes, uncovering new regulators of ribosome biogenesis (including CCDC86, ZNF236, and SPATA5L1), transcription (C7orf26), and mitochondrial respiration (TMEM242). In addition to assigning gene function, single-cell transcriptional phenotypes allow for in-depth dissection of complex cellular phenomena-from RNA processing to differentiation. We leverage this ability to systematically identify genetic drivers and consequences of aneuploidy and to discover an unanticipated layer of stress-specific regulation of the mitochondrial genome. Our information-rich genotype-phenotype map reveals a multidimensional portrait of gene and cellular function.
    Keywords:  CRISPR; Integrator complex; Perturb-seq; cell biology; chromosomal instability; genetic screens; genotype-phenotype map; mitochondrial genome stress response; single-cell RNA sequencing
  26. Nat Genet. 2022 Jun 06.
      Posttranslational modifications of histones (PTMs) are associated with specific chromatin and gene expression states1,2. Although studies in Drosophila melanogaster have revealed phenotypic associations between chromatin-modifying enzymes and their histone substrates, comparable studies in mammalian models do not exist3-5. Here, we use CRISPR base editing in mouse embryonic stem cells (mESCs) to address the regulatory role of lysine 27 of histone H3 (H3K27), a substrate for Polycomb repressive complex 2 (PRC2)-mediated methylation and CBP/EP300-mediated acetylation6,7. By generating pan-H3K27R (pK27R) mutant mESCs, where all 28 alleles of H3.1, H3.2 and H3.3 have been mutated, we demonstrate similarity in transcription patterns of genes and differentiation to PRC2-null mutants. Moreover, H3K27 acetylation is not essential for gene derepression linked to loss of H3K27 methylation, or de novo activation of genes during cell-fate transition to epiblast-like cells (EpiLCs). In conclusion, our results show that H3K27 is an essential substrate for PRC2 in mESCs, whereas other PTMs in addition to H3K27 acetylation are likely involved in mediating CBP/EP300 function. Our work demonstrates the feasibility of large-scale multicopy gene editing to interrogate histone PTM function in mammalian cells.
  27. Blood Adv. 2022 06 14. 6(11): 3386-3397
      Understanding the genomic and epigenetic mechanisms of drug resistance in pediatric acute lymphoblastic leukemia (ALL) is critical for further improvements in treatment outcomes. The role of transcriptomic response in conferring resistance to l-asparaginase (LASP) is poorly understood beyond asparagine synthetase (ASNS). We defined reproducible LASP response genes in LASP-resistant and LASP-sensitive ALL cell lines as well as primary leukemia samples from newly diagnosed patients. Defining target genes of the amino acid stress response-related transcription factor activating transcription factor 4 (ATF4) in ALL cell lines using chromatin immunoprecipitation sequencing (ChIP-seq) revealed 45% of genes that changed expression after LASP treatment were direct targets of the ATF4 transcription factor, and 34% of these genes harbored LASP-responsive ATF4 promoter binding events. SLC7A11 was found to be a response gene in cell lines and patient samples as well as a direct target of ATF4. SLC7A11 was also one of only 2.4% of LASP response genes with basal level gene expression that also correlated with LASP ex vivo resistance in primary leukemia cells. Experiments using chemical inhibition of SLC7A11 with sulfasalazine, gene overexpression, and partial gene knockout recapitulated LASP resistance or sensitivity in ALL cell lines. These findings show the importance of assessing changes in gene expression following treatment with an antileukemic agent for its association with drug resistance and highlight that many response genes may not differ in their basal expression in drug-resistant leukemia cells.
  28. Nature. 2022 Jun 08.
      Large-scale human genetic data1-3 have shown that cancer mutations display strong tissue-selectivity, but how this selectivity arises remains unclear. Here, using experimental models, functional genomics and analyses of patient samples, we demonstrate that the lineage transcription factor paired box 8 (PAX8) is required for oncogenic signalling by two common genetic alterations that cause clear cell renal cell carcinoma (ccRCC) in humans: the germline variant rs7948643 at 11q13.3 and somatic inactivation of the von Hippel-Lindau tumour suppressor (VHL)4-6. VHL loss, which is observed in about 90% of ccRCCs, can lead to hypoxia-inducible factor 2α (HIF2A) stabilization6,7. We show that HIF2A is preferentially recruited to PAX8-bound transcriptional enhancers, including a pro-tumorigenic cyclin D1 (CCND1) enhancer that is controlled by PAX8 and HIF2A. The ccRCC-protective allele C at rs7948643 inhibits PAX8 binding at this enhancer and downstream activation of CCND1 expression. Co-option of a PAX8-dependent physiological programme that supports the proliferation of normal renal epithelial cells is also required for MYC expression from the ccRCC metastasis-associated amplicons at 8q21.3-q24.3 (ref. 8). These results demonstrate that transcriptional lineage factors are essential for oncogenic signalling and that they mediate tissue-specific cancer risk associated with somatic and inherited genetic variants.