Myeloid Transformation Research Articles

Recruitment of MLL1 complex is essential for SETBP1 to induce myeloid transformation

SummaryAbnormal activation of SETBP1 due to overexpression or missense mutations occurs frequently in various myeloid neoplasms and associates with poor prognosis. Direct activation of Hoxa9/Hoxa10/Myb transcription by SETBP1 and its missense mutants is essential for their transforming capability; however, the underlying epigenetic mechanisms remain elusive. We found that both SETBP1 and its missense mutant SETBP1(D/N) directly interact with histone methyltransferase MLL1. Using a combination of ChIP-seq and RNA-seq analysis in primary hematopoietic stem and progenitor cells, we uncovered extensive overlap in their genomic occupancy and their cooperation in activating many oncogenic transcription factor genes including Hoxa9/Hoxa10/Myb and a large group of ribosomal protein genes. Genetic ablation of Mll1 as well as treatment with an inhibitor of the MLL1 complex OICR-9429 abrogated Setbp1/Setbp1(D/N)-induced transcriptional activation and transformation. Thus, the MLL1 complex plays a critical role in Setbp1-induced transcriptional activation and transformation and represents a promising target for treating myeloid neoplasms with SETBP1 activation.

Clonotype-Immunophenotype Relationships in TET2 and IDH-Mutant Myeloid Transformation

Large scale molecular profiling studies in AML patients have suggested that stepwise acquisition of somatic mutations is crucial in driving leukemic development. High variant allele frequency (VAF) mutations in epigenetic modifier genes, such as TET2 and IDH1/2, are thought to occur early in AML pathogenesis while oncogenic mutations with typically lower VAF mutations, including FLT3 and NRAS, are suggested to occur late in disease evolution. While bulk DNA sequencing has catalogued co-mutations found in individual AMLs, it cannot unveil the heterogeneity and composition of clones that makes up the disease. Elucidating the architecture and clone-specific molecular profiles at the single cell resolution will be key to understanding how sequential and/or parallel mutation acquisition drives myeloid transformation.To assess the clonal architecture of AML, we previously performed single cell DNA sequencing (scDNA seq) in 146 patients with myeloid malignancies. We have further identified specific mutational combinations driving clonal expansion in TET2- or IDH1/2- mutant AML samples. These studies suggest TET2 and IDH1/2 can cooperate to promote clonal expansion with DNMT3A and NPM1 (Figure 1A). However, TET2 or IDH1/2 mutant clones that acquired KRAS mutations underwent minimal clonal expansion, suggesting mutant-pair specific fitness alterations (Figure 1B). To further identify how co-mutational pairing impacted clonal fitness and differentiation, we integrated the scDNA platform with immunophenotypic profiling of 45 cell surface markers and analyzed new TET2- and IDH1/2- mutant AML samples (Figure 1C). We identified clone-specific differences in lineage markers depending on co-mutational partners. NPM1 co-mutant clones were enriched for more primitive markers (CD33), whereas NRAS co-mutant clones possessed high expression of myeloid differentiation markers (CD14/CD11b), suggestive of clone-specific fitness landscapes across hematopoietic differentiation. We also identified divergent clonotype-immunophenotype patterns in TET2- and IDH2-mutant clones harboring NPM1/RAS mutations, suggesting that initiating mutations may prime mutant clones for very different evolutionary trajectories as they acquire similar mutations in leukemogenesis (Figure 1D).To deterministically delineate the relationship between clonal evolution and myeloid transformation, we generated Cre-inducible single (Tet2 -/-), double (Tet2 -/-/Nras G12D and Tet2 -/-/Npm1 cA/wt), and triple (Tet2 -/-/Npm1 cA/wt/Nras G12D) mutant mice and evaluated differences in chimerism, immunophenotype, and survival. We observed a shortened survival for double and triple mutant mice, compared to Tet2 -/- only mice (Figure 1E). As previously reported, Tet2 -/-/Nras G12D mice developed a CMML-like phenotype. Critically, the addition of Npm1 resulted in a more rapid disease onset and transformation to AML (Figure 1F). Moreover, triple mutant WBM transplanted to form a fully penetrant disease into secondary recipients, while double mutant Tet2 -/-/Nras G12D WBM failed to form disease within 3 months of transplant, suggesting a difference in the cell population responsible for disease propagation. Immunophenotypic alterations were evident with Tet2 -/-/ Nras G12D displaying an increase in Mac1 +Gr1 + cells compared to Tet2 -/-/Npm1 cA/wt/Nras G12D mice which possessed increased Mac1 +Gr1 - cells and expansion of lineage negative cells (Figure 1G). These findings align with the clonotype specific expression patterns observed in clinical specimen and suggest that myeloid transformation and maturation biases are influenced by specific mutational combinations. [Display omitted] DisclosuresMiles: Mission Bio: Honoraria, Speakers Bureau. Bowman: Mission Bio: Honoraria, Speakers Bureau. Carroll: Janssen Pharmaceutical: Consultancy; Incyte Pharmaceuticals: Research Funding. Levine: Astellas: Consultancy; Janssen: Consultancy; Auron: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Incyte: Consultancy; Imago: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Research Funding; Prelude: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Lilly: Honoraria; Morphosys: Consultancy.

Treatment of Ropeginterferon Alpha-2b Achieves Hematologic Remission and Molecular Response in Patients with Hydroxyurea- and/or Anagrelide-Resistant/Intolerant Myeloproliferative Neoplasms

▪Background:Ropeginterferon alpha-2b (Ropeg) is a novel pegylated interferon-alpha approved for the treatment of polycythemia vera (PV) in Europe and Taiwan. Prior to its approval in Taiwan, the major options for patients with myeloproliferative neoplasms (MPNs) included hydroxyurea (HU) and/or anagrelide. Patients who are HU/anagrelide resistant/intolerant have limited options, as ruxolitinib is not subsidized by the national health insurance in Taiwan for PV. In 2017, the manufacturer provided a compassionate use program (CUP) for patients who were HU and/or anagrelide resistant/intolerant. Herein, we assessed the efficacy and safety of Ropeg in 20 MPN patients.Methods:To be eligible, patients must be resistant or intolerant to currently available therapies for MPN in Taiwan, mainly HU and anagrelide. Patients with autoimmune disorders, psychiatric illness, and acute/chronic infections were excluded. An accelerated dosing regimen was used, starting from 250 µg, and increased by 100 µg every two weeks until it reached the target dose of 500 µg, if no self-reported discomforts or abnormalities in biochemical or hematological profiles were observed. Efficacy assessments included hematologic parameters, phlebotomy need, and JAK2V617F allele burden. Hematologic remission was defined as platelets ≤400 x 10^9/L and white blood cells <9.5 x 10^9/L for essential thrombocythemia (ET), and platelets ≤400 x 10^9/L, white blood cells <10 x 10^9/L, and hematocrit <45% with no phlebotomy in the past 3 months for PV. Molecular response was defined as a reduction in JAK2V617F allele burden of at least 50% from baseline if baseline value was less than 50%, and a reduction of at least 25% from baseline if the baseline level was at least 50%. Each case was independently reviewed and approved for the use of Ropeg by both Institutional Review Board and the Ministry of Health and Welfare in Taiwan.Results:A total of 20 patients received treatment, which included 14 PV, 4 ET, 1 post-ET myelofibrosis (MF), and 1 pre-fibrotic primary MF (Table 1). There were 12 female and 8 male patients with a median age of 56.1 years old. Of these 20 patients, 18 had JAK2V617F mutation and 5 had a history of thrombosis. Of the 18 ET and PV patients, 13 achieved hematologic remission. The ET patients seemed to achieve hematologic remission faster than PV patients (19.3 vs. 33.2 weeks). Of the 18 patients with JAK2V617F mutation, 7 PV patients and 1 post-ET MF patient achieved molecular response, which took a median of 46 weeks after Ropeg treatment. Reduction in JAK2V617F allele burden was observed in 12 patients. One MF patient discontinued treatment due to disease progression. Another PV patient discontinued treatment due to acute myeloid leukemia transformation, although after treatment, the patient returned to PV state and continued Ropeg treatment. Overall, the drug was well tolerated, as most of the treatment-related adverse events (AEs) were mild to moderate. The AE profile was consistent with those from the phase 3 PROUD/CONTI-PV study. There were no unbearable side effects that led to treatment discontinuation.Conclusion:Our study provided evidence in the efficacy and safety of Ropeg for the treatment of HU-/anagrelide-resistant/intolerant MPNs. Hematologic remission was observed in ET and PV patients, whereas molecular response was observed in only PV patients, possibly due to the small sample size of ET patients. Our experience with Ropeg suggests it to be a promising option for the treatment of MPNs with drug-resistance/intolerance. [Display omitted] DisclosuresChen: PharmaEssentia: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen, Celgene, Novartis, and Panco Healthcare: Honoraria. Shih: PharmaEssentia Co: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Ltd: Research Funding; Ltd: Research Funding; Novartis: Research Funding. OffLabel Disclosure:Ropeginterferon alfa-2b is a novel interferon alpha indicated for the treatment of polycythemia vera in Europe and in Taiwan. This abstract describes the use of this agent for the treatment of myeloproliferative neoplasm patients with hydroxyurea/anagrelide resistance/intolerance.

The Study of Thrombosis in Polycythemia Vera

▪Objective In order to understand the clinical characteristics, disease progression and survival of polycythemia vera (PV) patients, explore the risk factors affecting thrombosis in PV patients, and establish a new prognostic integral model that is consistent with the characteristics of thrombosis in PV patients in China.Methods The clinical features, laboratory features, thrombosis, myelofibrosis transformation, leukemia transformation and survival of 975 patients with PV were studied retrospectively. By comparing the expected survival of a healthy Chinese population with age-sex matching, using the mortality rate of healthy Chinese as a reference, we calculated the standardized mortality ratio (SMR). The qualitative data were compared by the chi-square test or Fisher's test. ROC curve was used to determine the cutoff value of continuous variables, and Mann-Whitney U test and Kruskal-Wallis test were used for grouping comparison. The Kaplan-Meier method was used for survival analysis. Multivariate analysis was performed using the Cox regression model to analyze the risk factors of thrombosis and survival in patients with PV, and to establish a prognostic model for patients with PV.Results Results showed that among the 975 PV patients, 98.8% patients with JAK2 V617F mutation, 115 patients (11.8%) died, 202 patients (20.7%) developed secondary myelofibrosis, and 22 patients (2.3%) converted to acute myeloid leukemia. The median follow-up time of 975 patients with PV was 8(1 to 38) years in which the incidence of thrombosis was 5.4/100(95%CI[5.31/100,6.09/100]),the incidence of myelofibrosis transformation was 2.91(95%CI[1.45/100，5.15/100]), and the incidence of acute myeloid leukemia transformation was 0.28/100(95%CI[0.07/100,0.43/100]). A total of 339 patients (34.8%) had thrombosis, among which 93.2% (316/339) had arterial thrombosis and 20.1% (68/339) had venous thrombosis. Multivariate analysis shows that: Age ≥60 years old (P<0.0001, HR=3.824, 95%CI[1.930, 7.579]),DTA mutation (P=0.031, HR=1.953),95%CI[1.062，3.592 ])are risk factors for thrombosis in PV patients. According to the results of multivariate analysis, the risk model of PV thrombosis system (PTPSS) was established. In the low-risk group (0 points), the intermediate -risk group (1 points) and the high-risk group (≥2 points), the incidence of thrombosis in the high-risk group was 77.3%, 38.8% and 29.8%, respectively.The results of thrombosis survival analysis showed that: high-risk group patients (n=44;The median thrombosis-free survival was 6 years,95%CI[1.413; 10.587]), patients in the intermediate-risk group (n=304;The median thrombosis-free survival was 18 years.95%CI[10.971; 25.029]), patients in the low-risk group (n=627; The median thrombosis-free survival was 22 years.95%CI[16.797; 27.203]).Conclusions Thrombosis is one of the most common disease progression in PV patients, and the incidence of arterial thrombosis is significantly higher than that of venous thrombosis. Even after treatment, the incidence of thrombosis is still high. PTPSS can effectively predict thrombosis in PV patients, and is a more reasonable prognostic model for guiding the prevention and treatment of PV thrombosis. We will further explore the relationship between monocytes and inflammatory factors and thrombosis in patients with PV. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Recruitment of MLL1 Complex Is Essential for SETBP1 to Induce Myeloid Transformation

Abnormal activation of SETBP1 due to overexpression or missense mutations occurs frequently in various myeloid neoplasms and associates with poor prognosis. Direct activation of Hoxa9/Hoxa10/Myb transcription by SETBP1 and its missense mutants is essential for their transforming capability; however, the underlying mechanisms for such activation remain elusive. We found that knockdown of Mll1 in mouse myeloid progenitors immortalized by SETBP1 or its missense mutant SETBP1(D/N) caused significant reduction in the mRNA levels of Hoxa9/Hoxa10/Myb, suggesting that Mll1 is critical for their transcriptional activation induced by SETBP1 and its missense mutants. Physical association of MLL1 with SETBP1/SETBP1(D/N) was readily detected by co-immunoprecipitation in nuclear extracts of these cells, further suggesting that they may form a complex in myeloid cells to activate transcription. This complex formation is likely mediated by direct interactions between SETBP1/SETBP1(D/N) and MLL1 as both SETBP1 and SETBP1(D/N) are capable of interacting with multiple regions of MLL1 in binding assays using proteins synthesized by in vitro transcription and translation. To better understand the extent of SETBP1/SETBP1(D/N)-MLL1 interaction in regulating gene transcription, we carried out both ChIP-seq and RNA-seq analysis in mouse Lin -Sca-1 +c-Kit + (LSK) cells transduced by pMYs retrovirus expressing SETBP1 or SETBP1(D/N) or empty pMYs virus. These analyses revealed extensive overlap in genomic occupancy for MLL1 and SETBP1/SETBP1(D/N) and their cooperation in activating many oncogenic transcription factor genes in addition to Hoxa9/Hoxa10/Myb, including additional HoxA genes (Hoxa1, Hoxa3, Hoxa5, Hoxa6, and Hoxa7), Myc, Eya1, Mef2c, Meis1, Sox4, Mecom, and Lmo2. A large group of ribosomal protein genes were also found to be directly activated by MLL1 and SETBP1/SETBP1(D/N), identifying ribosomal biogenesis as another significant pathway induced by their cooperation. To further assess the requirement for MLL1 in SETBP1-induced transformation using a genetic approach, we also generated SETBP1/SETBP1(D/N)-induced immortalized myeloid progenitors and AMLs using LSK cells from Mll1 conditional knockout mice. Mll1 deletion in immortalized progenitors significantly decreased SETBP1/SETBP1(D/N)-induced transcriptional activation and their colony-forming potential. More importantly, Mll1 deletion significantly extended the survival of mice transplanted with SETBP1/SETBP1(D/N)-induced AMLs, indicating that Mll1 is essential for the maintenance of such leukemias in vivo. We further found that pharmacological inhibition of MLL1 complex using a WDR5 inhibitor OICR-9429 efficiently abrogated SETBP1/SETBP1(D/N)-induced transcriptional activation and transformation. Thus, MLL1 complex plays a critical role in Setbp1-induced transcriptional activation and transformation and represents a promising target for treating myeloid neoplasms with SETBP1 activation. DisclosuresMaciejewski: Novartis: Consultancy; Regeneron: Consultancy; Alexion: Consultancy; Bristol Myers Squibb/Celgene: Consultancy.

MDS-197: Magrolimab + Azacitidine Versus Azacitidine + Placebo in Untreated Higher-Risk (HR) Myelodysplastic Syndromes (MDS): The Phase 3, Randomized, ENHANCE Study

Context: Myelodysplastic syndromes (MDS) are myeloid clonal disorders characterized by cytopenia that primarily affect older individuals. Prognosis and treatment are guided by the Revised International Prognostic Scoring System (IPSS-R) criteria. Patients with intermediate-, high-, and very high-risk MDS (HR-MDS) have a median overall survival (OS) of 0.8–3.7 years. Despite the high unmet need for these patients, azacitidine (AZA) is the only approved therapy for HR-MDS that has improved OS in clinical trials to date. However, AZA monotherapy leads to low complete response (CR) rates with limited OS, indicating a need for alternative therapy. The monoclonal antibody magrolimab blocks the macrophage inhibitory immune checkpoint CD47, which is overexpressed on tumor cells. AZA increases expression of prophagocytic signals, facilitating synergy with magrolimab. In an ongoing phase 1b study, magrolimab + AZA led to high response rates and an acceptable safety profile without significant immune-related adverse events. Objective: To compare the efficacy and safety of magrolimab + AZA vs AZA + placebo in previously untreated patients with HR-MDS. Design: ENHANCE (NCT04313881) is a randomized, phase 3, double-blind, multicenter study. Interventions: Randomization is 1:1 to magrolimab + AZA or AZA + placebo with no crossover. Patients or Other Participants: Patients aged ≥18 years with previously untreated intermediate- to very high-risk MDS by IPSS-R are eligible. Magrolimab or placebo is administered intravenously with an initial 1 mg/kg priming dose to mitigate on-target anemia. An intrapatient dose-escalation regimen up to 30 mg/kg is administered through cycle 1, 30 mg/kg weekly dosing in cycle 2, with 30 mg/kg dosing every 2 weeks in cycle 3 and beyond. AZA is administered per regional prescribing information. Patients may remain on treatment until disease progression, relapse, loss of clinical benefit, or until unacceptable toxicities occur. Main Outcome Measures: The two primary efficacy endpoints are CR rate and OS. Secondary efficacy endpoints include red blood cell transfusion-independence rate, event-free survival, minimal residual disease-negative rate, time to acute myeloid leukemia transformation, and patient-reported Functional Assessment of Cancer Therapy-Anemia response rate. Results: Trial in progress. Conclusions: Patient enrollment began in September 2020; accrual is ongoing. Planned enrollment is approximately 520 patients globally. Funding Source: Gilead Sciences, Inc., Foster City, CA, USA.

Clonal hematopoiesis and associated diseases: A review of recent findings

Recent genome‐wide studies have revealed that aging or chronic inflammation can cause clonal expansion of cells in normal tissues. Clonal hematopoiesis has been the most intensively studied form of clonal expansion in the last decade. Clonal hematopoiesis of indeterminate potential (CHIP) is an age‐related phenomenon observed in elderly individuals with no history of hematological malignancy. The most frequently mutated genes in CHIP are DNMT3A, TET2, and ASXL1, which are associated with initiation of leukemia. Importantly, CHIP has been the focus of a number of studies because it is an independent risk factor for myeloid malignancy, cardiovascular disease (CVD), and all‐cause mortality. Animal models recapitulating human CHIP revealed that CHIP‐associated mutations alter the number and function of hematopoietic stem and progenitor cells (HSPCs) and promote leukemic transformation. Moreover, chronic inflammation caused by infection or aging confers a fitness advantage to the CHIP‐associated mutant HSPCs. Myeloid cells, such as macrophages with a CHIP‐associated mutation, accelerate chronic inflammation and are associated with increased levels of inflammatory cytokines. This positive feedback loop between CHIP and chronic inflammation promotes development of atherosclerosis and chronic heart failure and thereby increases the risk for CVD. Notably, HSPCs with a CHIP‐associated mutation may alter not only innate but also acquired immune cells. This suggests that CHIP is involved in the development of solid cancers or immune disorders, such as aplastic anemia. In this review, we provide an overview of recent findings on CHIP. We also discuss potential interventions for treating CHIP and preventing myeloid transformation and CVD progression.

Somatic Mutations Associated with Myeloid Transformation of RUNX1 -Mutated Familial Platelet Disorders with Propensity to Myeloid Malignancies Using Targeted Next-Generation Sequencing

INTRODUCTIONRUNX1 is a master transcription factor associated with hematopoiesis. Germ-line RUNX1 mutations are associated with familial thrombocytopenia and platelet disorders with bleeding phenotype and propensity to myeloid malignancies (FPDMM), such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Although only 40 families with germ-line RUNX1 mutations have been described, its autosomal dominant inheritance pattern and the finding that 50% of the families with more than one first degree relative with MDS and/or AML had RUNX1 mutations underscore the importance of this entity.Despite the elevated frequency of transformation to AML/MDS the array of somatic mutations involved in this process is not fully characterized. Here we describe three patients from two families who had a diagnosis of FPDMM, progressed to MDS and/or AML and had DNA samples available for Next-Generation Sequencing (NGS). METHODSWe identified five patients from two different family pedigrees with AML/MDS between 2014 and 2016. We've had access to DNA samples from three of these patients extracted from one of three sources (buccal swab, peripheral blood leukocytes, and bone marrow) throughout their follow-up. Patients agreed with sample collection after informed consent, according to the Declaration of Helsinki.NGS was performed using a custom-designed 48 gene panel related to myeloid malignancies, using the IonTorrent® sequencing platform as previously described (PMID: 28486043). RESULTSThe pedigrees of both families are described in Figure 1. Patient I-2 from pedigree 1 was diagnosed with acute lymphoblastic leukemia at age 57 and died shortly after. Her son, patient II-3 came to us initially with thrombocytopenia (80,000-100,000/ μl) and mild bleeding symptoms (bleeding assessment tool score: 3) 12 years prior to his AML diagnosis. He also had a colorectal cancer (treated surgically) diagnosed 6 months prior. At AML diagnosis, he had a normal karyotype and underwent standard induction chemotherapy and achieved a complete response. He received one cycle of consolidation with high-dose cytarabine and died shortly after due to metastatic colorectal cancer. His oral epithelial cell DNA sample from the beginning of follow-up was positive for a RUNX1 p.Arg166Ter mutation. Bone marrow (BM) DNA sample from the AML diagnosis showed an additional p.Arg204Ter RUNX1 mutation and an additional p.Gln139fs NFE2 mutation.Patients III-4 and III-5 from pedigree 2 belonged to a family with an extensive previous history of thrombocytopenia and AML. Patient III-5 came to us with thrombocytopenia (50,000-70,000/μl) 2 years prior his AML diagnosis. BM DNA obtained at this time was positive for a p.Arg201Ter RUNX1 mutation and for a p.Pro95Arg SRSF2 mutation. At AML diagnosis, his karyotype was normal and BM DNA was positive for a p.Gly12Asp NRAS mutation in addition to the aforementioned mutations. He underwent three cycles of induction and received a matched unrelated donor bone marrow transplant (BMT) while in refractory disease. He achieved a complete remission that lasted a year and is currently under treatment with hypomethylating agents. His oral epithelial cell DNA sample obtained prior his BMT was only positive for the RUNX1 p.Arg201Ter mutation.Patient III-4 had been followed up for thrombocytopenia for three years and presented to us with pancytopenia and multilineage dysplasia. BM DNA at the onset of pancytopenia showed a p.Arg201Ter RUNX 1 mutation, and emerging TET2 Ser471fs and NRAS Gly12Asp mutations. These findings are summarized in Table 1. CONCLUSIONSWe report a longitudinal DNA sequencing follow-up of three patients with FPDMM. Our findings highlight the heterogeneity of genetic pathways to myeloid transformation in FPDMM. Some of these, as loss of the functioning RUNX1, have already been described. However, here we report for the first time the association of NFE2 and NRAS mutations with myeloid transformation in FPDMM. [Display omitted] DisclosuresOzelo:Pfizer: Consultancy, Research Funding, Speakers Bureau; Roche: Consultancy, Speakers Bureau; CSL Behing: Consultancy; Shire: Consultancy, Research Funding, Speakers Bureau; Biogen: Consultancy, Research Funding, Speakers Bureau; Grifols: Speakers Bureau; Novo Nordisk: Consultancy, Research Funding, Speakers Bureau.

Activation Levels of b-Catenin Signaling in Osteoblasts Define a Threshold for the Development of Myeloid Neoplasia of Variable Severity

A growing body of evidence indicates that osteoblasts have a direct pathogenetic role in the development of myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) and transformation of MDS to AML. Models of osteoblast-induced MDS or AML (OIAML) include b-catenin activation in osteoblasts inducing AML in mice and seen in more than 30% of MDS and AML patients; deletion of Dicer1 in osteoblast progenitors leading to MDS in mice; Noonan syndrome mutation (PTPN11) in stromal cells driving MPN in mice; and Schwachman-Diamond syndrome mutation in stromal cells driving MDS in mice and predicting AML progression in patients. To further understand the mechanism of OIAML we examined whether a defined threshold of AML-inducing signal by osteoblasts is required for disease development. For this purpose we constitutively activated b-catenin in osteoblasts using three different lines of 2.3Col1 a 1-Cre transgenic mice characterized by different recombination efficiencies in bone. Line 1, in which recombination efficiency was 80%. Line 2 was the same original line in which after several inter-breedings we detected a reduction in recombination efficiency to 20%. Line3, which has 75% recombination efficiency. Activation of b-catenin in osteoblasts using Line 1, led to the development of MDS which rapidly transformed to full-blown AML characterized by multi-organ infiltration with myeloid blasts, hematopoietic deregulation and lethality by 6 weeks of age. In contrast, activation of b-catenin in osteoblasts using Line 2 did not lead to AML development. Mice remained healthy and survived until at least 10 months of age, the entire time they were observed. Activation of b-catenin in osteoblasts using Line 3 led to anemia, with the appearance of myeloid blasts in the blood, marrow and spleen and segmented neutrophils in blood. In addition to what was observed with Line 1, Pelger Huet neutrophils and Howell-Jolly bodies were detected in the blood. The latter are RBCs with inclusions of nuclear chromatin remnants (damaged chromosome fragments) indicating genomic instability and a dysfunctional spleen, unable to clear them from the circulation, due to dense infiltration with blasts and megakaryocytes. A spectrum of atypical immature monocytes with abundant pale blue agranular cytoplasm with vacuoles were observed in the blood. Taken together these features resemble characteristics of human MDS/CMML. Mice die between 4 and 14 weeks of age, when they undergo “blast crisis” indicating transformation to AML. To examine whether and how these observations relate to the human disease, we compared the levels of b-catenin activation in osteoblasts (calculated as a function % osteoblasts (Lin-/CD34-/RUNX2+ cells) expressing activated b-catenin) with the different MDS diseases subtypes as those are defined by the 2008 World Health Organization (WHO) classification system. We found that increased levels of b-catenin activation in osteoblasts correlate with the severity of the MDS type and with conversion to AML. Collectively, these observations suggest that a minimal activation level of a key leukemogenic signal from the stroma, is required to initiate cancer development, and provides mechanistic insight into the formation and progression of preleukemic stem cells in AML and the transformation of MDS to AML. DisclosuresRaza:Onconova Therapeutics: Research Funding, Speakers Bureau; Kura Oncology: Research Funding; Novartis: Speakers Bureau; Genoptix: Speakers Bureau; Janssen R&D: Research Funding; Celgene Inc.: Research Funding; Syros Pharmaceuticals: Research Funding.

Promising Results of a Phase 1/2 Clinical Trial of Ruxolitinib in Patients with Chronic Myelomonocytic Leukemia

▪Introduction: Chronic myelomonocytic leukemia (CMML) is a myeloid neoplasm characterized by GM-CSF hypersensitivity, marrow dysplasia, blood monocytosis, and a propensity for acute myeloid leukemia transformation. The overall survival is estimated at 34 months with no drug therapies known to improve survival. We previously reported results of a phase 1 study demonstrating that ruxolitinib, a JAK1/2 inhibitor, is safe and has clinical activity in CMML at doses up to 20mg BID. To confirm efficacy we performed a phase 2 clinical trial and here report the combined results of the phase 1 and 2 clinical studies. Methods: All CMML WHO subtypes were included without regard to previous therapy. Key exclusion criteria included an ANC < 0.25x103 c/dL and a platelet count < 35x103c/dL. The use of GM-CSF analogs was prohibited. The Kaplan-Meier method was used to estimate survival and log-rank test to determine statistical significance. Somatic mutations were identified at baseline using a 49 gene targeted next generation sequencing panel (RainDance Technologies) and a custom 46-plex Luminex assay was used to profile baseline cytokine levels (R&D). The study was registered at clinicaltrials.gov NCT01776723 Results: Between April 2015 and October 2016, 29 patients were enrolled in the phase 2 study resulting in a combined 49 CMML patients treated with ruxolitinib. In phase 2, 56 percent of patients had the proliferative subtype (MPN-CMML) defined by WBC >13x109/L and 28% had higher-risk disease by the Global MD Anderson Scoring System. Six patients (20%) received a prior hypomethylating agent and 85% of patients were WHO subtype CMML-1. Therapy-emergent toxicities were similar to those reported in phase 1. The mean duration of therapy was 219 days (range, 6-675 days). Three hematologic responses, including bi- or trilineage responses (2 HI-E, 2 HI-P, 1 HI-N), one marrow CR, and one partial bone marrow response per 2006 IWG criteria were observed. Six of 13 patients (46%) with splenomegaly had a 50% or greater reduction in spleen size by physical exam. When combining IWG and spleen responses, the overall response rate was 46% (n=11 of 24), including clinical benefit by MDS/MPN IWG response criteria. Using the Myeloproliferative Symptom Assessment Form (MPN-SAF) administered electronically daily, a reduction in total symptom score was noted. The median OS (mOS) from start of treatment was 28 months (95% CI 18-38), and, for CMML-MDS and CMML-MPN, was 32 months and 28 months respectively, (p=0.3). To evaluate whether ruxolitinib may have disease-modifying properties in CMML, analysis of mOS from time of diagnosis was performed comparing all ruxolitinib treated patients (n=49) to an historical dataset with over 1800 CMML patients (Padron et al 2015). The mOS at the time of diagnosis in CMML ruxolitinib-treated CMML patients was 69 months compared to 31 months in the historical CMML cohort (p=0.03). Adjusting for age, Global MD Anderson model, CMML WHO subtype, CMML-MPN, and the interval from diagnosis, treatment with ruxolitinib was associated with improved OS (HR 0.42, p <0.005). Although no mutation or class of mutations was associated with response, peripheral blood plasma levels of CXCL9 (p=0.02) and decreased levels of CCL5 (p=0.018) and RAGE (p=0.016) were statistically associated with higher likelihood of response. Conclusions: Ruxolitinib has promising activity in CMML patients with particular benefit in those with proliferative symptoms and may have disease-modifying activity. The MPN-SAF may be a useful tool to measure symptom burden in the context of ruxolitinib therapy for CMML. Overexpression of cytokines and soluble innate immune receptors is associated with a higher likelihood of response to ruxolitinib therapy. Further clinical study is warranted to validate the biomarkers of response and disease modifying capacity of ruxolitinib in CMML. DisclosuresPadron:Incyte: Honoraria, Research Funding. Lancet:Bio-Path Holdings: Consultancy; Celgene: Consultancy; Boehringer Ingelheim: Consultancy; Jazz Pharmaceuticals: Consultancy; BioSight: Consultancy; Erytech: Consultancy; Janssen: Consultancy; Pfizer: Other: Institutional research funding, Research Funding; Novartis: Consultancy. Mesa:Promedico: Research Funding; Ariad: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy; Gilead Sciences, Inc.: Research Funding; Celgene Corporation: Research Funding; Galena Biopharma, Inc.: Consultancy; Incyte Corporation: Research Funding; CTI BioPharma Corp.: Research Funding. Roboz:AbbVie, Agios, Amgen, Amphivena, Array Biopharma Inc., Astex, AstraZeneca, Celator, Celgene, Clovis Oncology, CTI BioPharma, Genoptix, Immune Pharmaceuticals, Janssen Pharmaceuticals, Juno, MedImmune, MEI Pharma, Novartis, Onconova, Pfizer, Roche Pharmace: Consultancy; Cellectis: Research Funding. Steensma:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Equity Ownership; Takeda: Consultancy; Celgene: Consultancy; Janssen: Consultancy, Research Funding; H3 Biosciences: Consultancy; Onconova: Consultancy; Pfizer: Consultancy. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees. Komrokji:Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria.

ASXL1 Mutation Change the Migration Ability in JAK2V617F Positive Cell Lines through SDF-1/CXCR4 Axis

ASXL1 plays a vital role in regulation of the histone methylation and acts as a tumor suppressor. Majority of ASXL1 mutations are frame-shift and nonsense mutations. The most frequent mutation is C.1934dupG (P.Gly646TrpfsX12) with a loss of PHD domain and truncated protein formed. Reduced normal ASXL1 protein expression promotes myeloid transformation. ASXL1 mutations change the transcription of certain oncogenes and result in dysfunction of tumor stem cells.We found that ASXL1 mutations were identified in 29.1% (32/110) patients with primary myelofibrosis (PMF). Moreover, JAK2V617F mutated patients with ASXL1 mutations were more likely to present splenomegaly (median LCM: 10 vs. 5 cm, P=0.023), while in JAK2V617F negative patients, the association was gone. There is evidence that SDF-1α/CXCR4 axis plays an important role in the adhesion of leukemic cells and bone marrow stromal cells. Abnormal CXCR4 expression may contribute to constitutive migration of CD34+ cells in PMF. Accordingly, we supposed that on the basis of JAK2V617F mutation, mutated ASXL1 acts on the SDF-1α/CXCR4 axis leading to the stem cell mobilization and splenomagly.To further investigate the molecular mechanism of ASXL1 mutation in patients with JAK2V617F, we performed short hairpin RNA (shRNA)-mediated stable knockdown of ASXL1 in JAK2V617F mutated UKE1 and SET2 cell lines and JAK2V617F negative HL-60 cell line. In JAK2V617F mutated UKE1 and SET2 cells, ASXL1 knockdown accompanied with the significant elevation of CXCR4 mRNA and protein levels (Figure 1A); while in HL-60 cell, ASXL1 knockdown accompanied with the significant decreased expression of CXCR4 (Figure 1A). Next, we tested the effect of ASXL1 expression on cell migration ability by using transwell migration assay. Our observation showed that ASXL1 knockdown cells showed obvious promotion in migration in JAK2V617F mutated UKE1 and SET2 cells (P=0.028 and P<0.001 ), and CXCR4 antagonist AMD3100 could disrupt this effect (Figure 1B), and down regulation of ASXL1 expression accompanied with declined migration ability in HL-60 cells (P=0.0025, Figure 1B). Flow cytometry also confirmed the increased membrane CXCR4 expression in JAK2V617F mutated UKE1 and SET2 cells. In order to further explore the mechanism of ASXL1 effect on cell migration, we performed mRNA expression microarrary and the results showed that the function of cell adhesion ability, cell-matrix adhesion and leukocyte migration ability were up regulated in ASXL1 knockdown SET2 cells (Figure 1C).In summary, our data demonstrated that ASXL1 knockdown in JAK2V617F mutated UKE1 and SET2 cells resulted in increased expression of CXCR4 and enhanced cell migration abilities, which may indicated that ASXL1 mutation may act on SDF-1/CXCR4 axis. We speculate that under the JAK2V617F mutated background, ASXL1 knockdown may promote the migration ability of tumor stem cells and formation of tumor microenvironment through acting on SDF-1/CXCR4 axis and result in splenomegaly and extramedullary hematopoiesis in PMF patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genetic Validation of the Palmitoylation/Depalmitoylation Cycle As a Drug Target in NRAS Mutant Hematologic Malignancies In Vivo

The C-terminal hypervariable region (HVR) of Ras proteins specifies post-translational modifications required for their subcellular localization and biologic function. The HVR of all four Ras isoforms terminates with a CAAX motif that is prenylated by farnesyltransferase, producing a weak membrane binding affinity that is further stabilized by a second signal motif. For K-Ras4b, this signal is provided by a polybasic lysine domain that allows direct localization of the protein at the plasma membrane (PM). By contrast, H-Ras, N-Ras, and K-Ras4a PM localization requires palmitoylation at cysteine(s) adjacent to the CAAX motif and is maintained through a dynamic cycle regulated by depalmitoylation and repalmitoylation. Importantly, these differences raise the provocative possibility that inhibiting palmitoylation could selectively target NRAS mutant cancer cells without toxicity to normal cells that retain intact K-Ras4b function.Indeed, Nras oncogenes with a cysteine-to-serine second site amino acid substitution at the palmitoylation site (C181S) exhibited defective transforming activity in hematopoietic stem and progenitor cells (HSPCs) ex vivo and in vivo (Cuiffo et al., Blood 2010; Xu et al., Blood 2012). However, these studies relied on retroviral promoters to express oncogenic N-RasG12D that resulted in supra-physiological protein levels in HSPCs. To overcome this limitation, we retargeted embryonic stem cells that were used to generate Lox-STOP-Lox (LSL) NrasG12D/+ knock in mice ( Haigis et al., Nat Genet 2008 ) to create a NrasG12D,C181S allele (hereafter called NrasC181S). We then performed intercrosses to generate Mx1-Cre, NrasC181S/C181S mice as well as cohorts of congenic Mx1-Cre, NrasWT/WT and Mx1-Cre, NrasG12D/G12D control animals. As expected (Li et al ., Blood 2011; Wang et al ., Cell Cycle 2011), Mx1-Cre, NrasG12D/G12D mice that were injected with poly(I:C) at weaning had a mean white blood cell (WBC) count of 58.3 x 103/µL at six months of age (Fig.1 A) and died from hematologic disease after 225 days (range 119-267 days, n= 14, Fig. 1B). By contrast, Mx1-Cre, NrasC181S/C181S mice had normal WBC counts at the same time point (mean: 11.34x103 WBC/µL) and remained well until 19-20 months of age (n=12). The difference in survival between these groups was highly significant (p=3x10-6 by Kaplan-Meier analysis). Detailed analysis of the hematopoietic compartment at six months of age (n=5 per genotype) revealed profound splenomegaly (mean weight: 1.272g) in NrasG12D/G12D mice, but normal spleen weights in the NrasWT/WT and NrasC181S/C181S animals (Fig. 1C). The expansion of granulocyte-monocyte progenitors (GMPs), megakaryocyte-erythroid progenitors (MEPs), and more primitive CD48+ Lin- c-Kit+ Sca1+ (LKS) cells previously observed in NrasG12D/G12D mice was also rescued by the C181S second site mutation. Furthermore, in vitro culture of HSPC-enriched bone marrow cells over a range of granulocyte-macrophage colony-stimulating factor (GM-CSF) concentrations revealed normal cytokine sensitivity of NrasC181S/C181S cells, while NrasG12D/G12D cells were characterized by cytokine-independent growth and GM-CSF hypersensitivity as previously described. Importantly, Ras-GTP levels were similarly elevated in bone marrow cells from both NrasG12D/G12D and NrasC181S/C181S mice compared to wild-type cells. This indicates that the C181S mutation abrogates the cytokine hypersensitivity and increased cell proliferation that results from oncogenic N-RasG12D expression without reverting the canonical biochemical consequence of the oncogenic G12D substitution. This observation, in turn, suggests that abnormal subcellular localization and trafficking of oncogenic N-Ras potently suppresses myeloid transformation. Taken together, these in vivo studies directly validate the palmitoylation/depalmitoylation cycle as a therapeutic target in hematologic malignancies and potentially in other cancers characterized by oncogenic NRAS mutations. [Display omitted] DisclosuresFirestone:Calico Life Sciences: Employment.

Loss of a 7q gene, CUX1, disrupts epigenetically driven DNA repair and drives therapy-related myeloid neoplasms

AbstractTherapy-related myeloid neoplasms (t-MNs) are high-risk late effects with poorly understood pathogenesis in cancer survivors. It has been postulated that, in some cases, hematopoietic stem and progenitor cells (HSPCs) harboring mutations are selected for by cytotoxic exposures and transform. Here, we evaluate this model in the context of deficiency of CUX1, a transcription factor encoded on chromosome 7q and deleted in half of t-MN cases. We report that CUX1 has a critical early role in the DNA repair process in HSPCs. Mechanistically, CUX1 recruits the histone methyltransferase EHMT2 to DNA breaks to promote downstream H3K9 and H3K27 methylation, phosphorylated ATM retention, subsequent γH2AX focus formation and propagation, and, ultimately, 53BP1 recruitment. Despite significant unrepaired DNA damage sustained in CUX1-deficient murine HSPCs after cytotoxic exposures, they continue to proliferate and expand, mimicking clonal hematopoiesis in patients postchemotherapy. As a consequence, preexisting CUX1 deficiency predisposes mice to highly penetrant and rapidly fatal therapy-related erythroleukemias. These findings establish the importance of epigenetic regulation of HSPC DNA repair and position CUX1 as a gatekeeper in myeloid transformation.

Clinicopathologic correlates and natural history of atypical chronic myeloid leukemia.

There are limited data on the clonal mechanisms underlying leukemogenesis, prognostic factors, and optimal therapy for atypical chronic myeloid leukemia (aCML). The authors evaluated the clinicopathologic features, outcomes, and responses to therapy of 65 patients with aCML. The median age was 67 years (range, 46-89 years). The most frequently mutated genes included ASXL1 (83%), SRSF2 (68%), and SETBP1 (58%). Mutations in SETBP1, SRSF2, TET2, and GATA2 appeared at variant allele frequencies (VAFs) greater than 40%, whereas other RAS pathway mutations were more likely to appear at low VAFs. The acquisition of new, previously undetectable mutations at transformation was observed in 63% of the evaluable patients, with the most common involving signaling pathway mutations. Hypomethylating agents (HMAs) were associated with the highest response rates but with a short duration of response (median, 2.7 months). Therapy with ruxolitinib was not associated with clinically significant responses as a single agent or in combination with an HMA. Allogeneic stem cell transplantation was the only therapy associated with improved outcomes (hazard ratio, 0.144; 95% CI, 0.035-0.593; P = .007). Age, platelet counts, bone marrow blast percentages, and serum lactate dehydrogenase (LDH) levels were independent predictors of survival and were integrated in a multivariable model that allowed the prediction of 1- and 3-year survival. aCML is characterized by high frequencies of ASXL1, SRSF2, and SETBP1 mutations and is associated with a high risk of acute myeloid leukemia transformation. Response and survival outcomes with current therapies remain poor. The incorporation of age, platelet counts, bone marrow blast percentages, and LDH levels can allow survival prediction, and allogeneic stem cell transplantation should be considered for all eligible patients.

Characteristics of Cohesin Mutation in Acute Myeloid Leukemia and Its Clinical Significance.

The occurrence of gene mutation is a major contributor to the initiation and propagation of acute myeloid leukemia (AML). Accumulating evidence suggests that genes encoding cohesin subunits have a high prevalence of mutations in AML, especially in the t(8;21) subtype. Therefore, it is important to understand how cohesin mutations contribute to leukemogenesis. However, the fundamental understanding of cohesin mutation in clonal expansion and myeloid transformation in hematopoietic cells remains ambiguous. Previous studies briefly introduced the cohesin mutation in AML; however, an in-depth summary of mutations in AML was not provided, and the correlation between cohesin and AML1-ETO in t (8;21) AML was also not analyzed. By summarizing the major findings regarding the cohesin mutation in AML, this review aims to define the characteristics of the cohesin complex mutation, identify its relationships with co-occurring gene mutations, assess its roles in clonal evolution, and discuss its potential for the prognosis of AML. In particular, we focus on the function of cohesin mutations in RUNX1-RUNX1T1 fusion.

Overexpression of Hmga2 activates Igf2bp2 and remodels transcriptional program of Tet2-deficient stem cells in myeloid transformation.

High Mobility Group AT-hook 2 (HMGA2) is a chromatin modifier and its overexpression has been found in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Level of Hmga2 expression is fine-tuned by Lin28b-Let-7 axis and Polycomb Repressive Complex 2, in which deletion of Ezh2 leads to activation of Hmga2 expression in hematopoietic stem cells. To elucidate the mechanisms by which the overexpression of HMGA2 helps transformation of stem cells harboring a driver mutation of TET2, we generated an Hmga2-expressing Tet2-deficient mouse model showing the progressive phenotypes of MDS and AML. The overexpression of Hmga2 remodeled the transcriptional program of Tet2-deficient stem and progenitor cells, leading to the impaired differentiation of myeloid cells. Furthermore, Hmga2 was bound to a proximal region of Igf2bp2 oncogene, and activated its transcription, leading to enhancing self-renewal of Tet2-deficient stem cells that was suppressed by inhibition of the DNA binding of Hmga2. These combinatory effects on the transcriptional program and cellular function were not redundant to those in Tet2-deficient cells. The present results elucidate that Hmga2 targets key oncogenic pathways during the transformation and highlight the Hmga2-Igf2bp2 axis as a potential target for therapeutic intervention.

HHEX promotes myeloid transformation in cooperation with mutant ASXL1

AbstractAdditional sex combs-like 1 (ASXL1), an epigenetic modulator, is frequently mutated in myeloid neoplasms. Recent analyses of mutant ASXL1 conditional knockin (ASXL1-MT-KI) mice suggested that ASXL1-MT alone is insufficient for myeloid transformation. In our previous study, we used retrovirus-mediated insertional mutagenesis, which exhibited the susceptibility of ASXL1-MT-KI hematopoietic cells to transform into myeloid leukemia cells. In this screening, we identified the hematopoietically expressed homeobox (HHEX) gene as one of the common retrovirus integration sites. In this study, we investigated the potential cooperation between ASXL1-MT and HHEX in myeloid leukemogenesis. Expression of HHEX enhanced proliferation of ASXL1-MT–expressing HSPCs by inhibiting apoptosis and blocking differentiation, whereas it showed only modest effect in normal HSPCs. Moreover, ASXL1-MT and HHEX accelerated the development of RUNX1-ETO9a and FLT3-ITD leukemia. Conversely, HHEX depletion profoundly attenuated the colony-forming activity and leukemogenicity of ASXL1-MT–expressing leukemia cells. Mechanistically, we identified MYB and ETV5 as downstream targets for ASXL1-MT and HHEX by using transcriptome and chromatin immunoprecipitation–next-generation sequencing analyses. Moreover, we found that expression of ASXL1-MT enhanced the binding of HHEX to the promoter loci of MYB or ETV5 via reducing H2AK119ub. Depletion of MYB or ETV5 induced apoptosis or differentiation in ASXL1-MT–expressing leukemia cells, respectively. In addition, ectopic expression of MYB or ETV5 reversed the reduced colony-forming activity of HHEX-depleted ASXL1-MT–expressing leukemia cells. These findings indicate that the HHEX-MYB/ETV5 axis promotes myeloid transformation in ASXL1-mutated preleukemia cells.

AML1-ETO inhibits acute myeloid leukemia immune escape by CD48

The t(8;21)(q22;q22) translocation is the most common chromosomal translocation in acute myeloid leukemia (AML), and it gives rise to acute myeloid gene 1 (AML1)-myeloid transforming gene 8 (ETO)-positive AML, which has a relatively favorable prognosis. CD48 is a favorable prognosis factor that is downregulated in AML patients. AML can escape immunosurveillance of natural killer (NK) cells by decreasing CD48 expression. The correlation between AML1-ETO and CD48-mediated immune evasion is not well understood. Here, we show that AML1-ETO can increase CD48 expression, which is regulated by AML1-ETO/P300-mediated acetylation. AML1-ETO can inhibit AML immune escape from NK cell recognition and killing by increasing CD48 expression. This study describes a novel mechanism by which AML1-ETO can inhibit AML immune escape by increasing CD48 acetylation, thereby providing new evidence about AML patients with AML1-ETO oncogene infusion having better clinical outcomes.

Myeloid transformation by MLL-ENL depends strictly on C/EBP.

Chromosomal rearrangements of the mixed-lineage leukemia gene MLL1 are the hallmark of infant acute leukemia. The granulocyte-macrophage progenitor state forms the epigenetic basis for myelomonocytic leukemia stemness and transformation by MLL-type oncoproteins. Previously, it was shown that the establishment of murine myelomonocytic MLL-ENL transformation, but not its maintenance, depends on the transcription factor C/EBPα, suggesting an epigenetic hit-and-run mechanism of MLL-driven oncogenesis. Here, we demonstrate that compound deletion of Cebpa/Cebpb almost entirely abrogated the growth and survival of MLL-ENL-transformed cells. Rare, slow-growing, and apoptosis-prone MLL-ENL-transformed escapees were recovered from compound Cebpa/Cebpb deletions. The escapees were uniformly characterized by high expression of the resident Cebpe gene, suggesting inferior functional compensation of C/EBPα/C/EBPβ deficiency by C/EBPε. Complementation was augmented by ectopic C/EBPβ expression and downstream activation of IGF1 that enhanced growth. Cebpe gene inactivation was accomplished only in the presence of complementing C/EBPβ, but not in its absence, confirming the Cebpe dependency of the Cebpa/Cebpb double knockouts. Our data show that MLL-transformed myeloid cells are dependent on C/EBPs during the initiation and maintenance of transformation.

Single-cell mutation analysis of clonal evolution in myeloid malignancies.

SummaryMyeloid malignancies, including acute myeloid leukemia (AML), arise from the expansion of hematopoietic stem/progenitor cells which acquire somatic mutations. Bulk molecular profiling suggests step-wise mutation acquisition, where mutant genes with high variant allele frequencies (VAFs) occur early in leukemogenesis and mutations with lower VAFs are thought to be acquired later1–3. Although bulk sequencing informs leukemia biology and prognostication, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate mutational order. To delineate the clonal framework of myeloid malignancies, we performed single cell mutational profiling on 146 samples from 123 patients. We found AML is dominated by a small number of clones, which frequently harbor co-occurring mutations in epigenetic regulators. Conversely, mutations in signaling genes often occur more than once in distinct subclones consistent with increasing clonal diversity. We next mapped clonal trajectories for each sample and uncovered mutation combinations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our studies of single cell clonal architecture provides novel insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression.