RUNX1 Mutations Research Articles

Salvage Autologous Peripheral Blood Stem Cell Transplantation after the Development of tMDS or AML in Multiple Myeloma Patients

Multiple Myeloma (MM) patients are often treated with high-dose chemotherapy and autologous stem cell transplantation (ASCT) for disease control. Lenalidomide is typically used as maintenance but results in an estimated 3.3% chance of developing a therapy-related myeloid neoplasm (TMN) typically myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML). This complication is often devastating with a median survival for those with a TMN of just 13 months. Over the past 6 years, 4 patients with multiple myeloma who developed a TMN were treated with a salvage ASCT using banked stem cells collected prior to their initial ASCT and before lenalidomide maintenance. None of these patients qualified for an allogeneic transplant. Melphalan 140 - 200 mg/m2 was used as conditioning prior to infusion of the peripheral blood stem cells, resulting in successful MDS/AML disease control. Case 1 = 61-year-old man with MM initially treated with CyBorD then transplanted in 2010. Post transplant treatment included lenalidomide maintenance, followed by a variety of salvage regimens over the next 7 years including thalidomide, carfilzomib, daratumumab and pomalidomide. He developed cytopenias evaluated by bone marrow biopsy in May 2017. This showed tMDS (Karyotype 47-48, XY, der (5)t(5:20)(q11.1;q11.1), +19, der(21:22)(q10:q10),+2~3mar[cp11]/46-48,idem,-der(5),+20[cp5]/46,XY[4]. FISH confirmed deletion 5q in 76% and loss of chromosome 5 in 6.5% of cells. NGS showed a frame shift mutation of TP53 at a variant allele frequency of 92.5%. He underwent a second ASCT in Aug 2017 and recovered normal hematopoiesis within three weeks. Unfortunately, he developed an aggressive relapse of his multiple myeloma 9 months later and succumbed from refractory multiple myeloma 14 months after the salvage transplant, without evidence of MDS. Case 2 = 72-year-old man with MM initially treated with CyBorD then an ASCT in May 2015. Lenalidomide maintenance was continued until October 2017 when he developed severe thrombocytopenia and circulating blasts. A bone marrow biopsy showed tMDS EB-1. Cytogenetics 46, XY t(3:21) (q26.2,q22) [18]/46, XY[2]. He received a second ASCT after melphalan 140 mg/m2 in Oct 2017 followed by recovery of normal blood counts. Bone marrow biopsy showed complete remission. He remains in remission from both the multiple myeloma and MDS almost 6 years later without any further therapy. Case 3 = 65-year-old man with MM treated with bortezomib + dexamethasone followed by ASCT in Aug 2012. He received maintenance lenalidomide for 4 years but then progressed. In Jan 2018 he developed pancytopenia and bone marrow biopsy demonstrated MDS with ringed sideroblasts. Cytogenetics were normal but NGS showed mutations of ASXL1, RUNX1, SF3B1, SETBP1 and DNMT3A. He underwent a second ASCT using his banked stem cells after melphalan 140 mg/m2 in Jan 2018 followed by full hematopoietic recovery. A repeat bone marrow biopsy in March 2022 showed persistent MDS harboring ASXL1, SF3B1, and DNMT3A mutations, but no detectable RUNX1 or SETBP1 abnormalities. His multiple myeloma remains in remission >5 years later. He receives only intermittent erythropoietin to maintain a Hgb of 11. Case 4 = 58-year-old man with MM initially treated with CyBorD followed by an ASCT in May 2014. He received lenalidomide maintenance but relapsed multiple times after 4 years ultimately complicated by renal failure requiring permanent dialysis. In March 2022 he developed pancytopenia. Bone marrow biopsy showed 15% blasts harboring del(5q), del(17p) and trisomy 8 by FISH. NGS demonstrated a mutation of ASXL1, and a copy number loss of TP53. Six months of decitabine did not improve his blood counts. In Dec 2022 he underwent a second ASCT after melphalan 140 mg/m2. A repeat bone marrow biopsy in Feb 2023 showed a complete morphological remission with a normal NGS. He is off all treatment 8 months post-transplant other than erythropoeitic support due to ongoing dialysis-dependent renal failure. The development of TMN is an uncommon but devastating event in patients with multiple myeloma after ASCT and lenalidomide maintenance. These patients responded well to a salvage ASCT using banked stem cells. This approach was well tolerated and provided durable hematopoietic recovery. Second ASCT using banked autologous stem cells should be considered in such patients who are not otherwise candidates for allogeneic transplantation.

The Significance of Variant Allele Frequency in SF3B1 Mutated Myelodysplastic Neoplasms/Syndromes

Introduction: Recent iterations of the World Health Organization (WHO) and international consensus classification (ICC) guidelines for myelodysplastic neoplasms (MDS) refined disease defining characteristics through incorporation of morphologic and genetic criteria. Despite these welcome updates, discrepancies between the WHO and ICC remain, particularly with respect to variant allele frequency (VAF) threshold in disease defining mutations such as SF3B1 and TP53. We evaluated patients with SF3B1-mutated MDS, a specific molecular class of MDS in both classification schemes , providing additional biologic and survival information to inform both diagnosis and prognosis. Methods: We queried the International Working Group for Prognosis in MDS (IWG-PM) cohort of adult (age ≥ 18 years) patients (pts) with MDS (N= 3,323; Bernard et al. NEJM Evidence 2022) for baseline clinical demographic, cytogenetic and molecular information, and clinical outcomes. SF3B1 VAF was assessed using targeted next generation sequencing. Demographics were analyzed using descriptive statistics. Between group differences were assessed using the Wilcoxon rank-sum test or Fisher's exact test as appropriate. Time to event endpoints were analyzed using the log-rank method. Multivariate analysis utilized cox proportional hazards regression. Results: We identified 447 (14%) pts met WHO 5th criteria for MDS with low blasts and SF3B1 mutation. Among these, 30 (7%) of pts had a SF3B1 VAF < 10% ( SF3B1low;currently classified as MDS, not otherwise specified by ICC) while 93% (N=417/447) had SF3B1 VAF of ≥ 10% ( SF3B1high). Median VAF in pts with SF3B1low vs. SF3B1high was 6% (range: 2.2-9.9%) vs. 37% (range: 10.9-54.1%). Pts with SF3B1low vs. SF3B1high were older (median age 79 vs. 74, p=0.010), and had lower median platelet (185 x10 9/L vs. 264 x10 9/L, p < 0.001) and white blood cell levels (3.94 x10 9/L vs. 5.46 x10 9/L , p < 0.001). The median number of mutations other than SF3B1 in SF3B1low and SF3B1high was 2 (range: 0-5 vs. 0-8, p=0.42), and SF3B1 was the sole genetic alteration in 10% vs. 20%, respectively (OR=2.2, p=0.24). Common SF3B1 variants in SF3B1low vs. SF3B1 high included p.K666 (27% vs. 10%, OR: 0.31, p=0.01) and p.K700 (50% vs. 59%, OR: 1.42, p=0.44). In pts with SF3B1low, single-hit mutations in TP53 (13% vs. 3%, OR: 0.21, p=0.021), SRSF2 (10% vs. 3%, OR: 0.25, p=0.06), and RUNX1 (7% vs. 4%, OR: 0.60, p=0.37) were more frequent (Fig. A). Other co-mutations had similar frequencies in SF3B1low vs. SF3B1high MDS: TET2 (33% vs. 42%, OR: 1.45, p=0.44), DNMT3A (23% vs. 27%, OR 1.22, p=0.83), ASXL1 (17% vs. 13%, OR: 0.71, p=0.57).The distribution of SF3B1 VAF, and observed patterns of co-mutations did not differ by sample type (peripheral blood or bone marrow). More pts with SF3B1low vs. SF3B1high (76% vs. 46%, p: 0.0011) had a subclonal SF3B1 mutation compared with other co-occurring mutations. In the 213 (48%) pts with subclonal SF3B1, the most frequent dominant mutations included TET2 (35%, N=75), DNMT3A (21%, N=45), and ASXL1 (4%, N=8). Among the top co-mutations in SF3B1low vs. SF3B1high, SF3B1low was more frequently subclonal to TP53 (50% vs. 0%, p=0.04), SRSF2 (33% vs. 0%, p=0.2), and RUNX1 (50% vs. 0%, p=0.1). As anticipated, SF3B1high cases were enriched for very-low/low risk IPSS-M subtypes (78% vs. 43%, OR: 2.9, p=0.011) compared to SF3B1low cases which had an increased frequency of moderate IPSS-M subtypes (30% vs. 14%, OR: 0.39, p=0.03; Fig.B). Median leukemia free survival (LFS; 5 vs. 6 years, p=0.10) was not significantly different between pts with SF3B1low vs. SF3B1high MDS. Median overall survival (OS; 5 vs. 6.2 years, p=0.04) was significantly shorter in pts with SF3B1low, however after adjusting for SF3B1 VAF, age, and IPSS-M score, only age (HR: 1.05 95% CI: 1.03-1.07, p < 0.0001), and increasing IPSS-M score (HR: 2.60 95% CI: 2.10-3.20, p< 0.0001) significantly associated with OS. Conclusion: Patients with MDS with SF3B1-mutation VAF < 10% are older and have an increased frequency of other clonally dominant co-mutations in adverse risk genes (i.e., TP53, SRSF2, RUNX1). Despite more adverse disease biology, LFS and OS were similar between pts with SF3B1low vs. SF3B1high with respect to SF3B1 VAF after accounting for age and IPSS-M score. Future diagnostic and prognostic schema could account for differences in SF3B1lowdisease biology through use of comprehensive prognostic tools such as the IPSS-M score.

DNA Damage Response Constrains Cell Growth and Drives Clonal Hematopoiesis in Telomere Biology Disorders

Telomere biology disorders (TBD) are inherited diseases caused by genetic defects in telomere maintenance. Patients with TBD have critically shortened telomeres, leading to bone marrow failure (BMF) and an increased risk of myeloid neoplasms (MN). We conducted a study to understand the genetic changes driving clonal evolution in TBD patients and their distinct mechanisms compared to normal aging and sporadic myelodysplastic syndrome (MDS). We analyzed acquired genetic alterations in hematopoietic cells from 105 TBD patients. The diagnosis of TBD was made using a combination of clinical history, germline mutation status, and telomere length. Germline mutations were identified in 84% of patients, a family history consistent with TBD was seen in 46%, and 76% of patients had lymphocyte telomere lengths (TL) <1st %ile for their age, while 22% fell between the 1st and 10th %ile. Complications observed in our cohort included BMF (42%), myeloid neoplasms (17%), solid tumors (17%), cirrhosis (14%), and interstitial lung disease (34%). Clonal hematopoiesis was detected in 44 of 105 patients (42%), including 8 of 27 (30%) patients 18 years or younger. Somatic genetic alterations primarily involved the DNA damage response (DDR) pathway, with ATM and TP53 being the most frequently mutated genes (11 ATM mutations in 5 of 80 patients; 5 TP53 mutations in 4 of 84 patients; Fig. 1). Mutations typical of age-related clonal hematopoiesis were seen in 4 patients (3 ASXL1, 1 TET2). Somatic reversion of TBD germline lesions was found in 7 patients. Additionally, 11 somatic mutations were detected in genes involved in DDR, telomere maintenance, and cell senescence, including KAT6B, PALB2, TELO2, TOP1, and RTEL1. MDS-associated gene mutations were found in 15 patients (18%), 8 in the setting of MN. The most common mutations affected spliceosome genes with U2AF1 p.S34F and SF3B1 being most notable, including 1 U2AF1 S34F identified in a 15-year-old. Mutations in NPM1, RUNX1, WT1, SF3B1, and FLT3 were identified only after malignant transformation. Acquired cytogenetic changes were observed in 16 of 87 evaluable patients (18%), 8 in the setting of MN, and included gain of 1q in 5 patients (5.7%). Two patients with TP53 mutations developed a complex karyotype (Fig 1). To understand selective pressures contributing to clonal hematopoiesis development in TBD, we analyzed transcriptional changes in bone marrow (n=2) and peripheral blood (n=1) mononuclear cells (MNCs) of 3 TBD patients with different germline lesions using single cell RNA sequencing (scRNASeq). MNCs from TBD patients exhibited significant differences compared to healthy controls, showing downregulation of MYC targets, reduced translation, cell cycle arrest, and upregulation of ATM-dependent DDR pathway and FAS pathway (Fig 2). We hypothesized that mutations in DDR pathway genes, particularly somatic ATM loss, enable TBD hematopoietic stem and progenitor cells to overcome telomere-induced DDR induction and premature senescence. Consistent with our hypothesis, low dose ATM inhibition in TBD patients' fibroblasts significantly enhanced growth rate of TERC-mutated and DKC1-mutated cells, whereas control fibroblasts were unaffected. Somatic mutations in ATM in our cohort were typically observed in patients with TERC-mutated TBD (3 of 5, 3 of 34 with TERC-mutated TBD), very low TL, and extra-hematopoietic manifestations. Of the 11 ATM mutations, 3 were predicted loss of function and 4 were missense kinase domain mutations, known or predicted to result in loss of kinase activity. Somatic ATM loss occurred both as heterozygous or compound heterozygous mutations. The development of ATM mutations alone was not causative of malignancy; however, 2 patients with somatic ATM mutations developed myeloid neoplasms after acquisition of additional mutations (subclonal mutation in NPM1 in one patient and clonal replacement with a TP53-mutant clone in another patient). In conclusion, our findings highlight the role of telomere dysfunction-induced DDR and cellular senescence in bone marrow failure and clonal selection in TBD. ATM loss selectively enhances growth of TBD cells by inhibiting DDR activation which may ameliorate marrow failure but has the potential to increase the risk of myeloid neoplasms after acquisition of additional genetic events. Future studies should explore the possibility of a therapeutic window involving very low doses of ATM inhibition in TBD.

Genetic Evolution from Chronic Myeloproliferative Neoplasms to Acute Myeloid Leukemia: An Analysis of Forty-Six Paired Samples

Background: Secondary acute myeloid leukemia (sAML) may arise from chronic myeloproliferative neoplasm (MPN). The genetic evolution pattern may be understood by paired sample analysis. Knowledge of genetic change in MPN/sAML is still limited and more extensive studies should be done. Patients and Methods: Forty-six paired samples (27 males) at diagnosis of MPN (12 polycythemia vera, 6 essential thrombocythemia, 25 primary myelofibrosis and 3 MPN-unclassified) and sAML transformation were available. Mutational analyses of 27 genes ( JAK2V617F, CALR, MPL, ASXL1, IDH1, IDH2, TET2, DNMT3A, EZH2, SRSF2, U2AF1, SF3B1, ZRSR2, SMC1, STAG2, SMC3, RUNX1, SETBP1, IKZF1, WT1, K-RAS, N-RAS, C-CBL, TP53, RAD21, BCOR and BCORL1) were performed. Mutations and their variant allele frequencies (VAF) were measured by next generation sequencing. Results: At baseline (MPN), the mutation number was 2 (maximal 5). Forty-five samples harbored driver gene mutations (31 JAK2V617F, 13 CALR and 1 MPL). Twenty-eight samples harbored at least one epigenetic regulator mutations (11 TET2, 8 DNMT3A,7 ASXL1, 5 EZH2, 2 IDH2, 1 IDH1). Twelve samples harbored spliceosome mutations which were mutually exclusive (6 SRSF2, 3 SF3B1, 2 U2AF1 1 ZRSR2). Other mutations were RUNX1, BCORL1 (n=2 each), CBL and BCOR (n=1 each). In sAML, the median mutation number was 3 (maximal 7). Loss of mutation was found for JAK2V617F (9/31), TET2 (1/11) and SRSF2 (1/6). Acquisition was most common for RUNX1 (n=9), followed by TP53, ASXL1 (n=5 each), K-RAS (n=4) , ZRSR2, STAG2 (n=3 each). The median number of acquired mutation was 1 (maximal 5). The frequencies of gene mutations are summarized in Figure 1A. The baseline VAF of JAK2V617F was high (>35%) in 80.6% (25/31). In progression to sAML, VAF was constant in 29.0% (9/31, VAF changes <10%), increased in 20.0% (9/31, increased VAF> 10%), decreased in 41.9% (13/31, decreased VAF>10%) (Figure 1B). The baseline VAF in CALR mutation was high (>35%) in 91.3% (12/13). VAF remained stable in 76.9% (10/13), and loss of CALR mutation was not observed (Figure 1B). TET2 mutations tended to be stable (7/16), but clonal expansion (5/16 of all mutation events at MPN), or acquisition (n=2) could be found during transformation to sAML. DNMT3A mutations remained stable (5/8), exhibited clonal expansion (2/8), or acquisition (n=1) in sAML progression. ASXL1 mutations remained stable (6/7), with clonal expansion (1/7), or acquisition (n=5) during MPN/sAML progression. All EZH2 mutants had clonal expansion (5/5) and one acquired in sAML (Figure 1B). We found that CALR mutationwas often the founding clone with high VAF at MPN phase, stable clone during sAML, and might acquire mutations in signaling pathways (1 CBL, 1 KRAS), epigenetic regulators (2 ASXL1, 1 IDH2), transcription factors (2 RUNX1, 1 BCOR), spliceosome (2 ZRSR2, 1 SF3B1), and cohesin complex (1 STAG2). JAK2 mutationwas often preceded by other ancestral mutations such as TET2, ASXL1, DNMT3A, EZH2, IDH2, SRSF2 or ZRSR2. During evolution, leukemia arose from non- JAK2 mutated clones with mutations in EZH2, DNMT3A, RUNX1, EZH2, SETBP1, or acquisition of TP53, SF3B1, BCOR and STAG2 mutations) in sAML. In outcome, the median time from MPN to sAML was 82 (range 3.7 to 338.5) months. Female gender, loss of JAK2V617F, presence of U2AF1 and absence of IDH1 were associated with shorter time to sAML transformation. No other genetic mutation in our gene panel or the number of mutations harbored by patients in our cohort had significant impact on time to sAML. Marked increase of VAF was observed for JAK2 (7/22) , DNMT3A (1/7) , IDH1 (2/3), IDH2 (1/3), TET2 (3/10) , EZH2 (4/5), C-CBL (1/1) and SRSF2 (1/4). Marked decrease of VAF was observed for JAK2 (4/22), MPL (1/1), DNMT3A (1/7) , ASXL1 (1/8), SF3B1 (2/3), SRSF2 (1/4) and U2AF1 (1/2). Multiple mutational clones were detected for TET2 (two clones in 4 samples and 3 clones in 1). Clonal expansion and reduction were observed in two pairs of samples upon sAML transformation. Conclusions: At the MPN stage, most patients had driver mutations with frequent co-mutations of epigenetic modifier or spliceosome genes. CALR mutant clones were stable while for JAK2, sAML often arose from clones without JAK2V617F which acquired RUNX1, TP53, ASXL1 and K-RAS. Research funding:Ministryof Science and Technology Council,Taiwan (NSTC 112-2314-B-182-055) and Ministry of Health and Welfare, Taiwan (112-TDU-B-222-124001)

Metabolic Signatures and Pathway Rewiring in TET2-Mutated Myeloid Neoplasia

Background. TET2 mutations ( TET2 MT) are the most frequently observed somatic lesions in myeloid neoplasms (MN). TET2 belongs to an Fe2+ and α-ketoglutarate (αKG) dependent DNA-dioxygenase family that progressively oxidizes 5-methyldeoxycytidine (5mC), leading to promoter and enhancer mCpG demethylation essential for efficient transcription. TET2 accounts for the majority of DNA dioxygenase activity in hematopoietic stem and progenitor cells (HSPCs), and following its loss, the resulting accumulation of 5mC leads to a loss of lineage plasticity and transcriptional repression of tumor suppressor genes, thereby establishing a volatile pre-neoplastic state. TET2 is one of the major consumers of αKG, a major cofactor produced by the tricarboxylic acid (TCA) cycle. Therefore, loss of TET2 creates a metabolic perturbation in HSPCs. However, the impact of TET2 loss on the metabolism of leukemic cells and its subsequent effects on malignant evolution remains unclear. Methods. We generated isogenic TET2 -/- ( TET2 ko) leukemic cell models in THP1 and K562 using CRISPR-Cas9 knockout. We then utilized targeted and untargeted metabolic analyses by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry in these models. We aimed to investigate the intricate metabolic changes associated with TET2 and TET2 MT. To validate our findings, we extended our studies to primary cells derived from MN patients with and without TET2 lesions. We performed extensive cellular metabolism studies using 13C isotope labelled glucose and glutamine. Additionally, levels of 5mC and its oxidation products, namely 5-hydroxymethyldeoxycytidine, 5-formyldeoxycytidine, and 5-carboxydeoxycytidine were documented for correlating epigenetic modifications with TET2 activities. Results. Targeted metabolic analysis uncovered profound impacts of TET2 MT on key metabolic pathways including the TCA cycle, glycolysis, glutamine metabolism, and nucleoside biosynthesis (Figure A). Distinct changes were observed in cells from different lineage backgrounds, suggesting diverse metabolic pathway rewiring specific to cell origin. Despite the divergence among lineages, loss of TET2 created common and unique metabolic signatures, highlighting their crucial role in sustaining the survival and clonal advantages specific to TET2 MT. Representative signature compounds and their relative quantities in TET2 wildtype (WT) and TET2 MT are depicted in Figure A. The targeted metabolic analysis revealed a significantly lower level of glutamine in TET2 MTcells, suggesting a potential preference for exhausting glutamine as a source of energy production. For validation, we conducted 13C metabolic flux analysis (13C-MFA) using glucose (U-13C, 99%) and L-glutamine (U-13C, 99%). 13C-MFA revealed distinct enrichment patterns (Figure B). In the TCA cycle, the dominant isotopologues receiving [U-13C]-glucose were M+2 for citrate and isocitrate. However, for aKG, succinate (SA) and fumarate (FA), the M+2s were significantly lower in TET2 -/- compared to WT, suggesting that the major source for these metabolites were rewired from glycolysis to glutamine metabolism in TET2 -/-. This was confirmed by [U-13C]-glutamine labeling where dominant isotopologues, M+5 for aKG and M+4 for SA, FA and malate, were significantly higher at 24-hour time point in in TET2 -/- compared to WT. Untargeted metabolic analyses from isogenic cells were used to identify metabolic features associated with TET2 loss. These metabolic features were also assayed in primary AML cells with TET2 MT (n=12) and without: RUNX1 (n=20), SF3B1 (n=11), IDH1 (n=10), IDH2 (n=13), FH (n=3), as well as healthy donors as controls (n=6). Based on the molecular features identified in the TET2 ko isogenic cells, MN patients with TET2, IDH1/2, and germline FH mutations clustered more closely, suggesting shared metabolic signatures among these groups. In contrast, patients with RUNX1 and SF3B1 mutations formed separate clusters, demonstrating distinct metabolic profiles associated with these genetic alterations. Conclusion. Our findings provide comprehensive and novel insights into the metabolic consequences of TET2 inactivation in MN. The identified metabolic signatures and rewiring of key pathways may hold promise as potential therapeutic targets and offer opportunities for tailored treatment approaches in TET2MT MN.

Sex-associated differences in frequencies and prognostic impact of recurrent genetic alterations in adult acute myeloid leukemia (Alliance, AMLCG)

Clinical outcome of patients with acute myeloid leukemia (AML) is associated with demographic and genetic features. Although the associations of acquired genetic alterations with patients’ sex have been recently analyzed, their impact on outcome of female and male patients has not yet been comprehensively assessed. We performed mutational profiling, cytogenetic and outcome analyses in 1726 adults with AML (749 female and 977 male) treated on frontline Alliance for Clinical Trials in Oncology protocols. A validation cohort comprised 465 women and 489 men treated on frontline protocols of the German AML Cooperative Group. Compared with men, women more often had normal karyotype, FLT3-ITD, DNMT3A, NPM1 and WT1 mutations and less often complex karyotype, ASXL1, SRSF2, U2AF1, RUNX1, or KIT mutations. More women were in the 2022 European LeukemiaNet intermediate-risk group and more men in adverse-risk group. We found sex differences in co-occurring mutation patterns and prognostic impact of select genetic alterations. The mutation-associated splicing events and gene-expression profiles also differed between sexes. In patients aged <60 years, SF3B1 mutations were male-specific adverse outcome prognosticators. We conclude that sex differences in AML-associated genetic alterations and mutation-specific differential splicing events highlight the importance of patients’ sex in analyses of AML biology and prognostication.

Genomic Characterization of Acute Myeloid Leukemia with Aberrations of Chromosome 7: A Multinational Cohort of 523 Patients

Introduction: Aberrations of chromosome 7 [abn(7)] are found in ≈ 10% of newly diagnosed acute myeloid leukemia (AML) and associate with a dismal prognosis. A large-scale comprehensive exploration of the underlying genetic heterogeneity in AML with abn(7) has yet to be performed and could add essential insights into the outcome of this poorly understood patient group. Methods: We collected diagnostic samples from 523 adult AML patients (median age 59 years) with abn(7). Whole-exome sequencing (WES) was performed to discover potentially underestimated genetic lesions in 61 paired diagnostic / remission samples. Subsequently, a gene panel including 66 genes and a SNP backbone for copy-number aberration (CNA) detection was designed and applied to the remaining 471 samples. The majority of patients (78%) were diagnosed with de novo (dn) AML, whereas 22% had secondary (s) or therapy-related (t) AML. Intensive induction treatment was administered to 80% of the patients, while 36 % underwent allogenic stem cell transplantation. Apart from 43% of patients with concomitant complex karyotype (abn(7)/CK+), 24% had -7 as a sole abnormality (-7 sole) and 13% del(7q) sole. Results: A mean of 15.3 single-nucleotide variants (SNVs) and 7.5 CNAs per patient were found by WES. Here, the most frequent recurrent SNVs were identified in TP53 (29.5%), followed by mutations (mut) in genes involved in epigenetic regulation ( DNMT3A, TET2, ASXL1, IDH2), transcription factors ( RUNX1), and genes affecting RAS-signaling ( NF1, KRAS), Figure 1. Targeted sequencing revealed 1829 SNVs with a VAF≥ 2% in 64 genes (mean: 3.8 SNVs / patient). 30% of patients harbored at least one mutation in genes located within the commonly deleted region of 7q, most frequently KMT2C, EZH2CUX1, SAMD9L, SAMD9, LUC7L2 and BRAF. The number of driver gene mutations was higher in CK- than in CK+ patients (4.5 vs. 3.3 SNVs). We found KRAS (OR 3.76, CI 1.17-16.87, P= .044) and RUNX1 (OR 3.61, CI 1.62-8.78, P= .003) mutations to be enriched in -7 sole patients, and FLT3 mutations to be associated with del(7q) sole status (OR 0.33, CI 0.13-0.82, P= .019). With respect to previously unknown lesions, a high amount of KMT2C mutations (16.6%) and recurrent alterations in FAT1 and TACC2 were discovered (6.4% each; Fig. 1). For KMT2C - located on chr7q36.1 - a total of 98 SNVs with mainly missense (73.5%) and truncating (20.4%) mutations at known cancer hotspots were noted. KMT2Cmut was associated with dnAML and AML with maturation morphology. In the entire cohort, the most common co-occurring chromosomal alterations discovered by high-resolution CNA analysis were deletions in chromosomes 5, 17, and 12. Our approach enabled the identification of small fragment deletions (≤10Mb) affecting the TP53, NF1, and ETV6 loci in 5-9% of all cases, which were missed by conventional G-banding. Cancer Cell Fraction and Bradley-Terry models were used to simulate the sequential order of genomic aberrations. While mutations in TP53 and epigenetic-related genes were early events, -7 and del(7q) were often subclonal and SNVs in NRAS, KMT2C very late events in leukemogenesis. Survival analyses in intensively-treated patients (n= 414) revealed that 61% reached complete remission, 67% relapsed, and median overall survival (OS) was 11.9 months. Abn TP53 and high WBC count were independently associated with shorter relapse-free survival (RFS). For OS, besides older age and high WBC count, we identified abn TP53, PTPN11mut, TET2mut,-5, and -18 as poor prognostic factors in multivariate analysis (Table 1). In contrast, IDH2mutconferred an independent favorable prognostic effect for RFS and OS. Notably, abn TP53 outcompeted the prognostic impact of CK+ (Table 1). Compared to TP53wt, patients with abn TP53/CK+ and abn TP53/CK- had a similar poor outcome with median RFS of 6 and 4 months (CK+/CK-, P&amp;lt;.001) and OS of 6.8 and 8.6 months (CK+/CK-, P&amp;lt;.001). In contrast to other genomic studies in myelodysplastic syndrome, we found abn TP53 to be associated with poor outcome irrespective of the single- or multihit mutation status following definitions of the latest ICC classification (Blood, 2022). Conclusion: Our results offer novel insights into the genomic landscape and clonal trajectory of AML with abn(7). This work unravels formerly underestimated genetic lesions ( KMT2Cmut) and alterations with high prognostic impact (abn TP53 and IDH2mut) for better future risk stratification.

Pre-Transplant Somatic Co-Occurring Mutations (by next generation sequencing) in Acute Myeloid Leukemia: Frequency and Impact on Clinical Outcomes after Allogeneic Hematopoietic Cell Transplantation - a Large Study on Behalf of the EBMT Acute Leukemia Working Party

Background: Acute myeloid leukemia (AML) is a very heterogeneous hematological malignancy, which includes numerous genetically defined subsets. The genomic classification of AML with the identification of mutations in transcription factors, epigenetic modifiers, spliceosome, cohesin complex, and signaling pathways has led to a more accurate risk stratification model. The main genetic aberrations included in the European LeukemiaNet (ELN) 2022 classification are NPM1 (risk group according to karyotype and FLT3-ITD status), FLT3-ITD (intermediate risk), bZIP in-frame mutated CEBPA (favorable risk), and RUNX1, ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, ZRSR2 and TP53 mutations (all belonging to the adverse risk group). In the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT), the frequency and prognostic value of different gene-gene interactions has not been studied and may differ from that of patients treated with chemotherapy alone. We aimed at evaluating, through the European Society for Blood and Marrow Transplantation (EBMT) database, the frequency and impact of different recurrent somatic mutations, either alone or in association, on prediction of relapse and survival in patients receiving allo-HSCT. Methods: This is a retrospective, registry-based, multicenter analysis from the EBMT with the approval of the EBMT Acute Leukemia Working Party. Adult patients aged more than 18 years with a diagnosis ofAML who received an allo-HSCT between 2013-2022, with an available genetic profile determined at diagnosis by next generation sequencing (NGS) were included. Results: We identified 836 allografted AML patients who had NGS performed at diagnosis. Most of these patients had de novo AML (88%), with a median age of 53 years (range: 18-78 years). Karyotype was favorable in 7%, intermediate in 69% and adverse in 24% of patients. At transplant, 74% of patients were in first remission (CR1) and 13% in CR2. The most frequent detectable mutations by frequency were DNMT3A (33%), FLT3 (29%), NPM1 (29%), TET2 (28%), NRAS (23%), RUNX1 (22%), WT1 (22%), BCOR (19%), ASXL1 (17%), IDH2 (17%), IDH1 (15%), SRSF2 (13%), STAG2 (12%), CEBPA (11%), TP53 (10%), KRAS (10%), and PTPN11 (10%) . By multiple correspondence analysis, two independent groups of co-occurring mutations were identified, the first group included DNMT3A, NPM1 and FLT3, the second group included ASXL1, SRSF2 and RUNX1. Outcome analysis was performed on the subset of 298 patients allografted in CR1 with available data for the aforementioned six genes ( DNMT3A, NPM1, FLT3, ASXL1, SRSF2 and RUNX1). Most of these patients had de novo AML (90%), with a median age of 53 years (range: 19-75 years). Patients received primarily reduced intensity conditioning (58%) and peripheral blood stem cells (93%) from matched sibling donors (35%), matched unrelated (28%), and haploidentical donors (21%). Seventy percent of these patients had intermediate-risk cytogenetics, while 27% were classified as adverse-risk. Median follow up calculated by the reverse Kaplan-Meier method was 2.5 years. Overall, the 2-year relapse incidence (RI), leukemia-free survival (LFS) and overall survival (OS) were 24%, 62% and 69%, respectively. When outcome analysis was performed according to the presence or absence of single mutations, none of the six mutations significantly affected RI or LFS. The 2-year OS was positively affected by the presence of NPM1 mutation (78% vs 66%; p=0.02) and FLT3 (79% vs 65%, p=0.01) but not significantly affected by the other 4 mutations. When mutations were investigated in groups, the 2-year RI, LFS and OS were 24%, 70% and 78%, respectively, for patients with NPM1 mutation regardless of other co-mutations, 35%, 56% and 69% for patients with FLT3-ITDand/or DNMT3A mutation, wild type NPM1, regardless of other co-mutations, 17%, 70% and 74% for patients with RUNX1 and/or ASXL1 and/or SRSF2 mutation without FLT3-ITD and with wild type NPM1 and wild type DNMT3A and 20%, 56% and 61% for patients with all six genes unmutated. Conclusion: NGS at diagnosis can be extremely useful in risk stratification of AML patients undergoing allo-HSCT, potentially allowing adequate post-transplant interventions. Notably, the 2-year LFS of 70% for patients harboring RUNX1 and/or ASXL1 and/or SRSF2 mutation indicates that allo-HSCT can overcome the adverse risk associated with these somatic mutations at diagnosis.

Phase Separation Mediates RUNX1- Mutation Leukemic Transformation

B ackground : The high degree of genetic heterogeneity in acute myeloid leukemia (AML) leads to refractory or relapse in some patients, so it is important to deepen the mechanistic studies and find specific drug targets.As a classical regulatory molecule associated with AML, the absence or down-regulation of RUNX1 can lead to the development of AML. Liquid-liquid phase separation is closely related to the occurrence and development of the disease, and it is not clear whether RUNX1 can participate in the pathogenesis of AML through phase separation. This study is the first to explore the involvement of RUNX1 in the pathogenesis of AML from the perspective of phase separation. Methods: The fluorescent expression of RUNX1 was detected by immunofluorescence. The extracellular phase separation of RUNX1 and the mutants were studied by prokaryotic expression of the target protein RUNX1. The intracellular phase separation of RUNX1 was studied by cellular transfection and live-cell imaging. The cellular differentiation ability after phase separation of AML cell lines was investigated by using Rachel Giemsa staining and flow cytometry. The specific mechanism of RUNX1 phase separation regulation of downstream target molecules was examined by Co-IP coupled with mass spectrometry and Western Blot. The effect of RUNX1 phase separation on the pathogenesis of AML was studied by using the AML mouse model. Results: In this study, we firstly confirmed that RUNX1 showed obvious spot aggregation during hematopoietic differentiation. We then confirmed that RUNX1 can perform liquid-liquid phase separation both in vitro and intracellularly. Furthermore, based on the structural domain of RUNX1 and the RUNX1 mutation sites in AML patients, it was confirmed that the IDR region of RUNX1 drove occurrence of phase separation, while the RHD region had an inhibitory effect on phase separation, and the RUNX1 point mutants associated with poor prognosis of AML had abnormal phase separation.The occurrence of phase separation of RUNX1 could regulate the differentiation function of AML cells, and the differentiation of AML cells in the phase separation-deficient type was inhibited. Transcriptome sequencing results showed that RUNX1 phase separation promoted the enrichment of signaling pathways such as cell differentiation, and it was also found that RUNX1 phase separation promoted downstream target molecules to participate in the regulation of AML cell function. In addition, we demonstrated that aberrant RUNX1 phase separation was involved in the pathogenesis of AML in the mouse model of AML. Conclusion: This study reveals for the first time that RUNX1 liquid-liquid phase separation is involved in the process of AML occurrence and development, and refines the functional regulation mechanism of RUNX1, providing new ideas for AML targeted therapy.

Clonal Evolution of Der(1;7)(q10;p10) Myeloid Neoplasms

Background der(1;7)(q10;p10) (der(1;7)) is a recurrent unbalanced translocation found in 1.5~6% of MDS and AML patients. It is caused by rearrangement of centromere alpha satellites in chromosomes 1 and 7 resulting in an amplification of 1q and a deletion of 7q. Previous studies comparing der(1;7)(+) cases to those with other lesions of 7q loss (-7/del(7q)) demonstrated a higher frequency of RUNX1 mutations, no TP53 mutations and a better prognosis for der(1;7) cases suggesting that der(1;7)(+) may define a unique entity of myeloid neoplasms (MN). However, with the lack of comprehensive molecular characterization using unbiased platforms such as whole genome/exome sequencing, the entire picture of der(1;7)(+) MN still remains unclear. Methods We enrolled a total of 148 MN cases harboring der(1;7) and an additional 3,238 der(1;7)(−) MN cases: -7/del(7q), +1q and ‘OTHER’. We first analyzed paired tumor/normal DNA from 26 der(1;7) cases using whole exome sequencing (WES), followed by targeted-capture sequencing of 329 genes, including those commonly mutated in MN and others newly identified in der(1;7) cases in WES. To investigate the origin of der(1;7)(+) MN, we also enrolled 11,234 healthy individuals to evaluate der(1;7) in clonal hematopoiesis, in which der(1;7) was detected on based on SNP array-based karyotyping. Results In accordance with previous reports, der(1;7) MDS cases showed an improved overall survival (OS) compared to -7/del(7q) MDS cases (Hazard Ratio (HR) =0.71, p=0.098), which however, was shorter than OS in +1q (HR=1.36, p=0.11) and ‘OTHER’ (HR=1.8, p&amp;lt;0.001) MDS cases. der(1;7)(+) cases were characterized by very high mortality from infection (45.5%), while mortality from disease progression was less common (36.4%), showing a sharp contrast to -7/del(7q) and ‘OTHER’ MDS cases which had 13.9% and 10.8% infection-related deaths, and 72.3% and 76.9% disease progression, respectively (Figure 1A.). WES in 26 der(1;7)(+) cases revealed a novel transcription factor, MYB in 2 cases, which was found in 18 (12%) of 148 der(1;7) cases using targeted-capture sequencing. der(1;7)(+) cases were characterized by high frequency of mutations in transcription factors, including RUNX1 (37.8%), GATA2 (12%), ETV6 (13%), and BCOR (14%) (Figure 1B.), which were significantly more frequent in der(1;7) cases than other sub-types (Odds Ratio (OR)&amp;lt;6)(Figure 1B.). Very high frequency of ETNK1 mutations is highly unique to der(1;7)(+) cases (18.9%) compared to that in other cases (&amp;lt;10%). High frequency of mutations in transcription factors seems to be a unique feature of der(1;7)(+) cases. Trisomy 8 and del(20q) CNAs were also common in der(1;7) cases (16% and 21%, respectively). Together with DNMT3A and del(20q), der(1;7) showed the highest variant allele frequency (VAF), suggesting the early origin of these lesions. Additionally, we identified der(1;7) in 6 out of 11,234 healthy individuals using SNP array-based highly sensitive copy number detection, of which 4 died from MDS, suggesting that der(1;7)(+) clonal hematopoiesis might be the origin of MDS in these cases. Moreover, we also analyzed longitudinal samples from 9 der(1;7)(+) cases using duplex-sequencing, der(1;7) showed consistently high VAF, further supporting that der(1;7) tended to represent the major clone. Conclusion Showing a unique set of co-mutation pattern and clinical features, der(1;7) MN defines a distinct subset of myeloid neoplasms, which may originate from der(1;7)(+) clonal hematopoiesis. der(1;7)(+) patients were at higher risk of life-threatening infection, which needs to be carefully prevented and monitored during their clinical course.

Conserved and Unique Regulatory Mechanisms of RUNX1 in Hematopoietic Stem/Progenitor Cell Subpopulations in Both Normal Hematopoiesis and FPD/AML Development

RUNX1 is a RUNT domain core-binding factor indispensable for the establishment of definitive hematopoiesis in vertebrate. In humans, heterozygous RUNX1 germline mutations are closely associated with Familial Platelet Disorder with propensity to develop AML (FPD/AML), a rare autosomal dominant disorder characterized by thrombocytopenia, platelet dysfunction, and markedly elevated risk for myelodysplastic syndrome and leukemia. RUNX1 regulates hematopoietic genes directly or indirectly, acting as a context-dependent activator or repressor of gene transcription. Here we have examined how RUNX1 regulates various subpopulations of hematopoietic stem/progenitor cells (HSPCs) to provide important insights into its physiologic and pathogenic roles and mechanisms.We used two complementary mouse models: a conditional RUNX1 knockout mouse, i,e, Mx-Cre;Runx1flox/flox, and a tetracycline-inducible FPD/AML patient derived RUNX1 S291fsX300 mutation knock-in crossed to a MLL-PTD knock-in mouse, i.e. MLL-PTD;RUNX1 S291fsX300. We monitored hematopoiesis and/or disease progression in both models by tracking CBCs in peripheral blood and analyzing bone marrow cells by flow cytometry. The bone marrow samples were sorted by flow cytometry to yield HSPC subpopulations: HSC (LSK CD34-Flt3-), GMP (LK FCRII/III+CD34+), and MEP (LK FCRII/III-CD34-). Cells were analyzed for transcriptome by deep RNA-seq, epigenetics by ATAC-seq, and gene targets by anti-RUNX1 CHIP-seq. Focusing on the HSC, GMP, and MEP subpopulations, we found that RUNX1 deletion in the hematopoietic system causes thrombocytopenia and HSC engraftment defects, and dramatic changes in HSPC subpopulations: RUNX1 deficiency resulted in an increase MegaK-primed HSCs via blocking their direct differentiation to megakaryocytes leading to thrombocytopenia, and caused a marked increase of GMPs and a significant decrease of MEPs. Induction of RUNX1 S291fsX300 mutant in MLL-PTD;RUNX1 S291fsX300 mice mimicked that of RUNX1 deletion, resulting similarly in thrombocytopenia, an expansion of Mega-HSCs, increased GMPs, and decreased MEPs. “Correction” of the RUNX1S291fsX300 mutation by doxycycline withdraw reversed the FPD phenotype as well as the cellular distribution changes in HSCs, GMPs and MEPs. RNA-seq analyses of DEGs found that RUNX1 acts both as an activator and a suppressor in gene transcription in HSPCs. In HSCs, among 1879 DEGs, 608 are upregulated and 1271 are downregulated in Runx1 -/- compared with WT cells. In GMPs, 2042 DEGs were seen, among which 908 are upregulated and 1134 are downregulated whereas in MEPs, 316 transcripts were upregulated and 1214 were downregulated amongst 1530 DEGs in Runx1 -/- cells. Combining the DEGs from RNA-seq with annotated ATAC-seq peaks, we discovered through gene ontology analyses that a set of systemic development genes are commonly regulated by RUNX1 via chromosome accessibility in all HSPC subpopulations, with potentially shared targets such as Jam2, Bcar1, Irf4 Insrr , Cxcl12, Tnfsf4, Rap1gap, Aldh1a1. In contrast, in HSCs, a unique set of hemostasis/platelet regulatory genes such as Itga2b, Gp5, Pdgfb and Vwf were seen as potential targets, consistent with our recent findings ( Blood Adv 7:2590). In GMPs, RUNX1 appears to specifically regulate inflammatory and innate immunity genes with potential targets such as Gfi1b, Ets1, Tnfrsf11a while in MEP, RUNX1 specifically regulates cell motility and migration gene sets affecting targets such as Notch1, Ifitm1, Hoxa7, Cd300a Cxcl10 Fut7, suggesting a role of RUNX1 in modulating MEP movement/location in the bone marrow. RUNX1 CHIP-seq analyses are under way to further clarify the potential targets in each HSPC subpopulation. Our studies indicate that RUNX1 regulates common as well as distinct transcriptional events in various HSPC subpopulations by context-dependent activation and suppression mechanisms. RUNX1 plays a universal role in the systemic developmental program of various HSPC subpopulations but appears to engage in unique lineage specific factors to deliver subpopulation-specific functions such as mega-K cell fate-priming of HSCs, inflammatory program in GMPs, and bone marrow location signals in MEPs. Ongoing analyses of the FPD/AML mouse genomic data in this context are expected to reveal contributions of patient RUNX1 mutations to the disease development.

Efficacy and Safety Analysis of Venetoclax Combined with Azacitidine in Real-World Treatment of Patients with AML

Objective: To evaluate overall survival (OS), response rate, time to first response, duration of response, depth of response, and safety in real-world treatment with Venetoclax (Ven) combined with Azacitidine (AZA) in patients with Acute Myeloid Leukemia (AML). Methods: From January 2019 to December 2021, 31 patients with AML who completed at least 2 courses of Ven (100-400mg, 28 days), the Ven dose was adjusted based on drug interactions particularly with azole antifungal prophylaxis, and AZA (75mg/m 2,7 days). Baseline characteristics: Median age 65 years (30-82 years), including 5 patients ≥75 years. ECOG score: 19 patients (61.3%) scored 0-1, 12 patients (38.7%) scored 2-3. Type of AML: 20 De novo cases (64.5%), 8 MDS or MPN conversion cases (25.8%), 3 primary AML-MRC cases (9.7%). Previous treatment: 22 patients (71%) were initially treated, 8 patients (25.8%) were relapsed/refractory, and 1 patient (3.2%) was maintained after remission with standard therapy. At baseline, grade 3 or greater cytopenia was associated: 3 cases were anemia (9.7%), 13 cases were neutropenia (41.9%), and 14 cases were thrombocytopenia (45.2%). Bone marrow original cell count: &amp;lt; 30% (10 cases), 30-50%(9 cases), ≥50% (12 cases). Cytogenetic risk: 1 case was low risk (3.2%), 19 cases were medium risk (61.3%), 10 cases were high risk (32.3%), and 1 case was no dividing phase (3.2%). Molecular mutations included: NRAS/KRAS mutation in 12 cases, DNMT3A mutation in 11 cases, NPM1 mutation in 9 cases, IDH1/2 mutation in 6 cases, FLT3-ITD mutation in 5 cases each, HOX11 positive, ASXL1, RUNX1 and NPM1+FLT3-ITD mutation in 3 cases each. Results: After a median of 3 courses of treatment (C2-C15), 3 of the patients underwent bridge hematopoietic stem cell transplantation after remission. The best efficacy was evaluated in CR+CRi: 22 patients (71%), PR: 5 patients (3 of whom were refractory to recurrence), SD: 3 patients (1 of whom was refractory to recurrence), and NR: 1 patient (refractory to recurrence). First-line efficacy was better than second-line or multi-line. Median time to start treatment to reach CR/CRi: 45 days, DOR: 314 days. The negative response rate of flow MRD in CR/CRi patients was 81.8%, including 11 cases of C1 conversion, 3 cases of C2 conversion, 3 cases of C3 conversion and 1 case of C4 conversion. Flow minimal residual disease (MRD) persisted in AML1-ETO subgroup. The early combined response rate of patients with important gene mutations: ASXL1 and RUNX1 mutation subgroup 100%, NPM1 mutation subgroup 77.78%, IDH1/2 mutation subgroup 83.33%, FLT3-ITD mutation subgroup 80%, HOX11 positive subgroup 80%, 66.67% in the NPM1+FLT3-ITD and NRAS/KRAS mutation subgroups, 53.55% in the DNMT3A mutation subgroup, and 1 patient with TP53 mutation was not in remission. The median OS of the first-line/ maintenance treatment group was 19 months, which was better than the median OS of the second-line or multi-line treatment group was 7 months. Safety: The incidence of tumolysis syndrome and early treatment-related mortality was 0; The incidence of early reduction or treatment interruption was higher (25.8%). Severe adverse reactions included sepsis in 2 cases, severe pneumonia in 2 cases and septic shock in 1 case. Conclusions: In the real world first-line treatment of high-risk AML patients with Ven combined with AZA, the disease control and survival were initially improved, and the early combined response rate (CR+CRi) of molecular subgroups IDH1/2, NPM1, RUNX1, ASXL1, FLT3-ITD and HOX11 were higher. The higher incidence of adverse events in early treatment and delayed treatment may be related to 41.9% of patients with grade 3 neutropenia at baseline, partly AML-MRC. So, based on Ven-28d/AZA, We still need to explore new therapeutic modalities which should balance the efficacy and safety.

Ubiquitin Ligase SCF-FBXO11 Controls a Network of RNA-Binding Proteins and Splicing in MDS

The F-Box protein FBXO11 is a substrate receptor of Skp-Cullin-F-Box (SCF) ubiquitin ligase complexes. Loss-of-function mutations in FBXO11 are associated with diffuse large B-cell lymphoma, and FBXO11 has been shown to regulate epigenetic drivers of erythroid cell maturation. In our prior study, we found that deletion of FBXO11 improved cell survival under cytokine deprivation in an MDS cell line. Furthermore, we had observed a striking decrease in FBXO11 protein levels in AML patient samples. However, how FBXO11 contributes to myeloid malignancies is unclear. Here, using RNA-sequencing, proteomics, and CRISPR-Cas9 screening, we reveal broad ubiquitylation of RNA-binding proteins (RBPs) and alternative splicing as a mechanism by which deregulation of FBXO11 enhances malignant cell function. In addition, we report the presence of mutations in the uncharacterized N-terminal extension of FBXO11 in both lymphoid and myeloid malignancies. We first observed that heterozygous deletion of FBXO11 increases colony-forming ability of MDS-AML cell lines in vitro whilea2-fold increase in expression results in decreased cell growth and apoptosis. In a transplant model with a retrovirally-driven Runx1 mutation, heterozygous deletion of Fbxo11 exacerbated neutropenia and shortened overall survival. However, depletion of FBXO11 in healthy human and murine HSPCs significantly impaired myeloid progenitor colony-formation, indicating a unique requirement for FBXO11 in myelopoiesis. Based on its tumor suppressive activity, we evaluated whole-exome sequencing data from patient samples of both lymphoid and myeloid malignancies for FBXO11 mutations. This analysis uncovered rare mutations in FBXO11 in myeloid malignancies and revealed a substantial cluster of previously unidentified mutations in the Q-rich, N-terminal extension of FBXO11. We found that this uncharacterized, extended protein isoform is predominantly expressed in both normal and malignant hematopoietic cells. In colony-forming assays, expression of the long isoform and several of the N-terminal mutations inhibited CD34+ progenitor function. On a cellular level, lack of this N-terminal extension in the short isoform resulted in aberrant subcellular localization of FBXO11 within the nucleus. To elucidate the mechanism of FBXO11 in myeloid malignancy, we performed immunoprecipitation of FBXO11 followed by proteomics to identify endogenous FBXO11 complexes in MDS-AML cells. We integrated these data with ubiquitin proteomics of WT and FBXO11-KO MDS cells to identify candidate substrates of SCF-FBXO11-mediated ubiquitylation. FBXO11 complexes are significantly enriched for RNA-binding proteins affecting mRNA splicing, a process commonly deregulated in MDS. FBXO11-KO MDS cells displayed broad depletion of ubiquitylation in a network of RBPs. As expected, targeting FBXO11 did not affect the level of transcripts of interacting proteins; however, there were variable changes in total protein levels indicative of post-transcriptional regulation. We then applied a focused CRISPR-Cas9-KO screen of candidate SCF-FBXO11 substrates in MDS-AML cells depleted of FBXO11. We observed significant bi-directional effects in colony-forming ability mediated by several of the FBXO11-regulated RBPs including TRIM28, HNRNPU, NPM1, and SYNCRIP. Though FBXO11 is significantly decreased in MDS compared to healthy controls, we saw no difference in FBXO11 expression between splicing factor WT and mutant samples, suggesting that the function of FBXO11 in regulating mRNA splicing in MDS is independent of these mutations. Compared to healthy controls, FBXO11-low MDS samples exhibited a greater number of alternative splicing events, predominantly in skipped or mutually-exclusive exons. To experimentally test whether FBXO11 indeed affects RNA splicing, we utilized a bichromatic splicing reporter assay in MDS-AML cells which showed that depletion of FBXO11 is sufficient to alter splicing of the reading frame in the cassette exon, read out as a shift from GFP to RFP. Finally, STRING protein network analysis revealed that the alternative splicing events occurring in FBXO11-low MDS patient samples affect key nodes in protein translation and metabolism. Collectively, our data support a model whereby the change in FBXO11-mediated ubiquitylation of RBPs drives alternative exon usage that exacerbates malignant cell function in MDS.

A Single-Center Large Cohort of Chronic Neutropenia Patients and a Model for Estimation of Congenital Neutropenias

Chronic neutropenia (CrN) is defined as neutropenia lasting longer than 3 months and has various underlying etiologies, including congenital neutropenia (CN). We aimed to determine the underlying etiologies of patients with CrN and define the characteristics at diagnosis suggestive for CN as a final diagnosis. The study included 197 pediatric and adolescent patients who were diagnosed with CrN between 2010 and 2022 in a single-center. Patients with transient neutropenia, splenomegaly, alloimmune, secondary autoimmune, myelodysplastic syndrome and hematological malignancies, other bone marrow failure syndromes such as Fanconi anemia, and aplastic anemia due to CrN were excluded from the study. The patients included in the study were sub-classified into 5 groups according to their final diagnosis. Group 1, patients with CN (n=64, %32.5) included all genetically verified CrN patients. Group 2, patients with primary autoimmune neutropenia (pIN) group (n=10, %5.1) with anti-granulocyte antibody (AGA) positivity. Group 3, patients with unclassified chronic idiopathic neutropenia (UCIN) (n=73, %37.1). Group 3 included patients who had a clinical severity resembling immune neutropenia, but whose AGA testing was either negative or not available; but the CrN resolved during follow-up. Group 4, included the patients with unclassified chronic benign neutropenia (UCBN) (n=34, %17.3) and included the patients with ongoing neutropenia, who do not have a history of serious infections, no underlying genetic cause for neutropenia, no AGA positivity. Group 5, unspecified congenital neutropenia (UCN) group (n=16, %8,1), was defined as the group of patients whose genetic tests did not detect a mutation in genes related to CN, but history reveales severe infections and a possible CN as an underlying cause. Genetic testing of patients varies from single gene evaluation with Sanger sequencing analysis to Whole Exome Sequencing (WES). The median age at diagnosis of neutropenia of the patients included in the study was 12 (0-168) months 53.8% of the patients were male (n=106). There was consanguinity between parents in 34.5% and at least 1 family member was diagnosed with neutropenia in the in 18.8%. Among the patients in Group 1, HAX-1 mutation was detected in 19 (29.7%) patients and it was found to be the most common genetic defect causing CN. Other common genetic disorders include ELANE mutations (18.8%), Shwachman Diamond Syndrome (10.9%), Hermansky Pudlak Syndrome (7.8%) and glycogen storage type 1 b (6.3%). G6PC3 deficiency, VPS45 deficiency, Barth syndrome and Kabuki syndrome in 2 each (3.1%), ADA2 deficiency, Cohen Syndrome, Poikiloderma with neutropenia, Prolidase deficiency, CLPB deficiency, Majeed syndrome, GINS4 deficiency, reticular dysgenesis, germline RUNX1 mutation were detected in 1 patient. There are 3 patients with acute myeloid leukemia and all of them died before hematopoietic stem cell transplantation (HSCT). Two of these patients were diagnosed with SDS, and one had ELANE mutation. One patient with the HAX-1 mutation developed non-Hodgkin lymphoma and was treated with HSCT and is still alive. There were 7 patients (3.6%) who underwent HSCT. 9 patients (4.6%) died. Except for 3 patients who died from AML and 1 who died after HSCT; 1 patient with Barth syndrome deceased related to associated dilated cardiomyopathy, Another patient with G6PC3 mutation died after complications of cardiac surgery due to associated cardiac anomaly and 3 patients (2 VPS45, 1 CLPB) died due to infections. Patients in Group1 and Group 2+Group3 were compared, in order to answer the question of “Can we create a model that predicts CN?”. Many parameters were compared and a multivariate model was created with statistically significant risk factors. CN was seen 6,3 times more in those with parental consanguinity, 8,9 times in those with a family history of neutropenia, 4,8 times in those with more than 2 hospitalizations due to infection, and 4,1 times more in those with recurrent oral involvement (p&amp;lt;0.05). Patients diagnosed with CN were estimated based on the results of the multifactor logistic regression model. The rate of correct identification of the patients by the model is 88.9%. In conclusion, our study is one of the largest single-center studies and suggests a novel classification of CrN patients.

RUNX1 Germline Mutations of Autosomal-Dominant Familial Platelet Disorder Are More Common Than Expected in Adult Myeloid Leukemias Indicating Highly Variable Penetrance

Germline (GL) RUNX1 (RUNX1GL) mutations result in autosomal-dominant familial platelet disorder (FPD) and are associated with heightened risk of myeloid neoplasia (MN). Indeed about 43% of RUNX1GL carriers are expected to develop MN, by the age of 50 yrs. 1 AML progression can be accompanied by biallelic RUNX1 hits. The phenotypic heterogeneity, incomplete penetrance, and leukemogenic risk depend on mutational burden and functional impact (topology of mutations, configuration) of RUNX1 and thus evolution of MN may be delayed. 2-3 We hypothesized that a large systematic study of RUNX1GL including types and topology will elucidate the impact of various RUNX1mutations ( RUNX1MT), their configuration, and molecular associations to provide prognostic insights and mechanistic conclusions as to the pathogenesis of these mutations. We analyzed over 9000 patients from Cleveland Clinic Foundation (CCF; n=7953) and public series. 4-5 We devised a novel rational algorithm to assess of RUNX1GL variants ( Fig1A). Stringent criteria were used that prioritized exclusion of false positives: GL benign and likely benign alterations were not included, while pathogenic and likely pathogenic (P/LP) variants were further curated. Carriers of VUS RUNX1MT that displayed past medical history or family history suspicious for FPD or leukemia were classified as VUS of suspicious pathogenicity (sVUS). The remaining variants, including P/LP, were further filtered and clinically annotated ( Fig1B). GL confirmation was possible in 22 alterations, e.g., wherein our algorithm resulted in ambivalent assignment. We found 627 RUNX1 variants (GL and somatic) in 557 patients, of which 8 were sVUS and 106 (17%) were found to be GL P/LP including 3 new RUNX1GL alterations (Y281*, S141L, S410*). The mean age at presentation of RUNX1GL carriers was lower (64 yrs.) compared to patients with somatic RUNX1MT(70 yrs., P= .0053). There was no difference in disease phenotype (AML, MDS, MDS/MPN) between GL, somatic, and biallelic (somatic/somatic) RUNX1MT carriers. Of note, 1 patient with predicted RUNX1GL variants had aplastic anemia. Cytogenetic analysis revealed that 68% of RUNX1GL cases were associated with normal karyotype. Four RUNX1GL cases also displayed trisomy 21. FLT3 ITD/PTDwas more prevalent in RUNX1GL carriers (14.8%) than in those with somatic RUNX1MT(4.7%) ( P= .021). PHF6 MT were more frequent in carriers of RUNX1GL (18%) compared to somatic RUNX1 carriers (9%) ( P= .078). Patients with a second RUNX1 hit included somatic biallelic (n=52)and GL/somatic (n=6) RUNX1 MT. The most common mutations co-occuring with biallelic RUNX1 hit werethose in EZH2 (27%, P= .025), ZRSR2 (19%, P= .020), U2AF1 (21%, P= .030) and PTPN11 (16%, P= .013) compared to monoallelic RUNX1MT. We further categorized RUNX1MT according to their predicted functional effects as: i) haploinsufficient (RUNT-truncating mutations); ii) dominant negative (post-RUNT nonsense, C-terminal frameshift, and missense mutations previously described as dominant negative); and iii) RUNT missense mutations. 6-7 We analyzed 93 (25%) mutations classified as haploinsufficient, 98 (27%) as dominant negative, and 134 (36%) as RUNT missense. There was no difference in the topology between somatic and GL hits. Survival analysis indicated that there was no difference in prognosis between dominant negative, haploinsufficient, and RUNT missense mutations ( P= .59). Among RUNX1GL carriers, there was also no prognostic difference between subtypes of RUNX1GL ( P= .896). To determine whether GL mutation types convey a greater penetrance, we compared the age of patients at presentation for somatic and GL. Specifically, carries of GL dominant negative mutations were diagnosed at a younger median age (60.5 yrs.) compared to carriers of GL haploinsufficient (68 yrs.) and RUNT missense (69 yrs.). However, this trend was not statistically significant. To rule out the effect of RUNX1GL lesions on genome instability, we quantified the mutational burden per each GL variant. Compared to RUNT missense, haploinsufficient GL variants were associated with a greater number of co-occurring mutations ( P= .013) while dominant negative GL variants were not ( P= .054). In summary, our study of the topological features of RUNX1GL variants showed that dominant negative RUNX1 mutations are frequent in MN and are not associated with specific phenotypes, but rather define a variable penetrance.

High Frequency of RUNX1 Mutation in Acute Myeloid Leukemia at Puerto Rico

Acute Myeloid Leukemia (AML) is the most common form of acute leukemia in adults, with a median age at diagnosis of 68 years. Recurrent genomic abnormalities are established as diagnostic, prognostic markers, and play an essential role in leukemogenesis. For instance, loss-of-function mutations of the runt-related transcription factor-1 (RUNX1) have been associated with distinct clinical and genetic features and overall adverse risk in adult AML. Lack of a specific targeted therapy and resistance to standard intensive chemotherapy likely accounts for the poorer outcome observed in AML expressing RUNX1. Additionally, germline RUNX1 mutations result in the well-described autosomal-dominant familial platelet disorder with predisposition to hematologic malignancies. Previuos studies has identified mutant RUNX1 in 5-10% of patients at a median age of 68 years old. The objective of our retrospective study was to identify the incidence and characteristics of RUNX1 mutations in our population. RUNX1 plays an important role in the regulation and differentiation of normal hematopoietic stem cells. Therefore, intragenic mutation of RUNX1 leads to impaired hematopoiesis and ultimately leukemia. The study examined newly diagnosed (ND) patients-AML with RUNX1 mutation by NGS; that were at least 18 years of age or older with cytogenetic and molecular analysis in a period of 4 years (2019-2022). According to the Puerto Rico Cancer Registry from 2015 to 2019 a total of 551 cases of AML were reported. All data regarding demographic, bone marrow biopsy, cytogenetics, and molecular markers were obtained from electronic medical record at San Juan City Hospital with institutional review board approval in acordance with the Declaration of Helsinki. The San Juan City Hospital is the major referral center on the island for initial evaluation and management of AML. A total of 157 patients with ND AML were identified during the study period. 58% female and 42% male with a median age of 57 years old at time of diagnosis. Compared to an expected ~5-10% incidence of RUNX1 mutations, our study population revealed the presence of RUNX1 in 24% of our AML patients, with a median age less than 60 years old. More specificially within the 37 patients with RUNX1 mutations, 46% female and 54% male with a median age of 56 years old with an average variable allele frequency(VAF) of 40%. Complex cytogenetics was identified in 41% of RUNX1 cases. Co-occuring mutation of RUNX1 with TP53 was identified in 19% of patients. Our study showed a high expression of RUNX1 in Puerto Rican ND-AML. The median age at diagnosis and the average VAF suggest the possibility of a germline RUNX1 in our population. Previous studies in medical literature have identified a correlation for germline RUNX1 in patients diagnosed before 60 years old and with a VAF above 40%. Future studies with larger sample size and longer follow-up may confirm if there is a germline predisposition in our patients for RUNX1.

Overcoming Venetoclax (Ven) Resistance through Glutamine (Gln) Depletion: Final Analysis of the Phase 1 Trial of Ven and Pegcrisantaspase (PegC) Combination in Relapsed and Refractory (R/R) Acute Myeloid Leukemia (AML)

Background: Ven-based therapies have been an advancement in the front-line treatment of AML; however development of resistance is common and the efficacy of Ven-containing regimens in R/R AML has been far less promising. We reported in vitro and in vivo depletion of Gln induced by long-acting Erwinia asparaginase, PegC, inhibited proliferation of complex karyotype (CK) AML and synergistically enhanced the antiapoptotic activity of Ven-mediated antagonism of Bcl-2 by decreasing the expression of proteins such as Mcl-1, whose translation is cap-dependent. Here we report the final results of our phase 1 clinical trial (NCT04666649) of VenPegC for treatment of R/R AML including patients (pts) previously treated with Ven. Methods: Pts received Ven 400 mg on D1-28 and PegC (500, 750, 1000 IU/m 2 in cohorts 1-3, IV) D1 &amp; D15 every 4 weeks (wk). Primary study endpoints were incidences of regimen limiting toxicities (RLT) and RP2D. Secondary endpoints included rate of composite complete remissions. Correlative studies included monitoring plasma amino acid levels and asparaginase activity, and expression of proteins regulating cap-dependent mRNA translation in bone marrow (BM) mononuclear cells pre- and post-VenPegC treatment. Results: As of July 31, 2023, 26 pts were enrolled of whom 24 received at least 1 dose of PegC and 17 completed at least 1 cycle of PegC (2 doses). The median age was 61 (range 24-79), 16 (62%) were female; 9 (35%) had CK-AML and 5 (19%) had TP53 mutations; 18 (69%) were previously treated with Ven. Median prior lines of treatment were 2 (range 1-5). No RLT was observed in cohort 1 (n=3). Cohort 2 was expanded to 6 pts due to transient asymptomatic hypertriglyceridemia. Two pts in cohort 3 (n=3) developed grade 3 hyperbilirubinemia determined as RLT. Expansion cohort included 11 pts who were treated at MTD/RP2D (PegC 750 IU/m 2 plus Ven 400 mg) given as out-pt without a need for hospitalization. No pancreatitis, thrombohemorrhagic events, or silent inactivation were observed in the entire study population. Overall response rate (ORR) for pts who received ≥1 cycle of PegC was 47% (8/17), Fig 1. CR+ CRh+CRi rate was 29% (5/17); 1 pt had a partial remission with full count recovery and is currently receiving cycle 6; 2 pts had stable disease. Four pts showed hematologic improvement. Twelve (71%) pts who received ≥1 cycle of PegC were previously treated with Ven and were Ven-refractory. ORR in Ven-refractory pts was 42% (5/12); of the 5 pts who harbored a RAS or PTPN11 mutation and previously treated with Ven, 60% responded. Remarkably, all pts (3/3) who had a RUNX1 mutation achieved a CR; Pt 6 (relapsed AML with NRAS/cKIT/RUNX1 mutations) achieved an MRD-negative CRh and underwent an allogeneic stem cell transplant (alloHSCT); Pt 12 (primary refractory AML with TP53/NRAS/RUNX1 mutations) achieved a clearance of BM blasts after 1 dose of PegC and achieved MRD-negative CR at the end of cycle 1, and Pt 15 (post-alloHSCT relapsed AML with NRAS/PTPN11/CSF3R/RUNX1 mutations) achieved a CRi and received 10 cycles of VenPegC. Pt 31 (post-alloHSCT relapsed AML with FLT3-ITD) who had persistent disease after gilteritinib achieved an MRD-negative CRi, pending 2 nd alloHSCT. Nadir plasma asparaginase activity of &amp;gt;0.1 IU/mL was achieved in cycle 1 for all pts who received ≥1 cycle of VenPegC. All pts with available results achieved undetectable asparagine (Asn) level by day 7 and Asn remained undetectable throughout the study. Plasma Gln reduction from baseline occurred in all pts (except 1) within cycle 1. Pts in cohort 1 reached nadir Gln after initial PegC exposure in an average of 22 days compared to 16 days in cohort 2. In general, pts who achieved CR had the lowest absolute plasma Gln levels. Available Glu and Gln are shown in Fig.1 for pts who achieved a CR/PR. Out of the 8 assessed pts (4 responders &amp; 4 non-responders), VenPegC inhibited cap-dependent translation after 1 cycle in responders, as evidenced by decreased phosphorylated eIF4E (active, pheIF4E) in 4/4 pts and decreased phosphorylated 4EBP1 (inactive, ph4EBP1), a negative regulator of eIF4E, in 2/4 pts. Conversely, pheIF4E and ph4EBP1 expression was increased in 3/4 and 4/4 of the non-responders, respectively (Fig.2). Conclusion: VenPegC is a novel regimen that can induce complete remission in some heavily pretreated R/R AML patients, even with previous exposure to Ven. Pts with RUNX1 mutation, whose translation depends on pheIF4E, may benefit further from this regimen.

A Proposal for a Classification of Ccus, MDS and AML Primarily Based on Genetic Abnormalities Considering the Biological Continuum of These Entities

Background: According to the 5 th edition of the WHO classification (WHO 2022) and the International Consensus Classification (ICC) the main parameters used for the definition of CCUS, MDS and AML are cytopenias, dysplasia, blast count and distinct genetic abnormalities. These entities represent a disease continuum as CCUS can progress to MDS and MDS to AML. Thus, we asked the question whether this continuum can be reflected in a proposed comprehensive classification system. Methods: We evaluated 222 CCUS, 718 de novo MDS and 751 de novo AML. The diagnosis was established following WHO 2022. Bone marrow samples were analyzed by cytomorphology, chromosome banding analysis, targeted NGS panel sequencing (median coverage 1500x) and WGS (median coverage 100x). Overall survival (OS) was available for 1552/1691 patients. Median follow-up with respect to OS was CCUS: 1.7 years, MDS: 9.3 years, AML: 6.7 years. Results: In the WHO 2022 classification a subset of AML entities (AML with defining genetic abnormalities (DGA)) is defined by fusions ( PML:: RARA, RUNX1:: RUNX1T1, CBFB:: MYH11, DEK:: NUP214, RBM15:: MRTFA other defined genetic alterations/ ODGA), rearrangements ( KMT2A, NUP98, MECOM) and distinct mutations ( NPM1, CEBPA). These well-established distinct entities were kept in the classification proposal. 511 AML cases (67%) were classified as AML DGA (Figure 1). The remaining 240 AML cases classified as AML myelodysplasia-related and AML defined by differentiation according to WHO 2022 were combined with MDS and CCUS cases and categorized hierarchically into 5 distinct groups based on the presence of the following genetic abnormalities (GA group): 1. 5q deletion, 2. SF3B1 mutation, 3. other spliceosome mutations (SP: SRSF2, U2AF1, ZRSR2), 4. IDH1/2 or DNMT3A or TET2 mutations (I/D/T), 5. None of these (NO) (Figure 1). Next, within these GA groups the presence of progression markers (PM) was evaluated: PM subset A: presence of a) biallelic TP53 alterations ( TP53bi), or b) mutations in RUNX1 or ASXL1 and mutations in RAS pathway genes or KMT2A-PTD (R/A+RAS/PTD); PM subset B: presence of a) complex karyotype without TP53bi (complex), or b) RUNX1,or ASXL1 mutations (R/A), or RAS pathway mutations or KMT2A-PTD (RAS/PTD); PM subset C: absence of these PM. The assignment into GA groups and PM subsets differed substantially between CCUS, MDS and AML. With respect to GA group CCUS, MDS and AML cases were assigned into GA groups del(5q): CCUS (0%), MDS (76%), AML (24%); SF3B1: CCUS (4%), MDS (91%), AML (5%); SP: CCUS (16%), MDS (55%), AML (29%); I/D/T: CCUS (45%), MDS (30%), AML (25%); NO: CCUS (27%), MDS (62%), AML (11%). With respect to PM subsets CCUS, MDS and AML cases were assigned into A: CCUS (1%), MDS (43%), AML (56%); B: CCUS (10%), MDS (54%), AML (36%); C: CCUS (27%), MDS (69%), AML (4%) (Figure 1). Next, OS was evaluated. Median OS for PM subsets A (n=168), B (n=301) and C (n=572) was 0.7, 2.0 and 6.8 years ( p&amp;lt;0.001), respectively and thus were named acute myeloid neoplasm (MN), high risk MN and low risk MN. Median OS in years in each GA group in acute MN was del(5q): 0.3, SF3B1: 0.7, SP: 1.1, I/D/T: 0.9 and NO: 0.3. The respective data for high risk and low risk MN were: 1.9, 3.3, 2.0, 1.5, 1.3 and 6.4, 7.4, 6.9, 5.8, and 6.8 years (Figure 1). Conclusions: The proposed classification aims at reflecting the biological continuum of CCUS, MDS and AML and allows reclassification during the course of the disease without changing the disease entity following the concept of MPN with chronic phase and blast phase. Further, the classification reflects prognosis and also directly indicates potential therapeutic targets. It is flexible as new genetic markers can easily be added without changing the overall concept.

Novel Agents with Efficacy Against Cellular Models of Familial Platelet Disorder with Myeloid Malignancy (FPD-MM) Associated with Germline Mutant RUNX1

RUNX1 is a master-transcriptional regulator involved in normal and malignant hematopoiesis. Germline, mono-allelic, missense mutations in RUNX1 cause large deletions or truncations that are mostly loss-of-function mutations. They cause familial platelet disorder (RUNX1-FPD), which evolves into myeloid malignancy (FPD-MM), e.g., MDS or AML. In addition to mutant (mt) RUNX1, FPD-MM cells commonly harbor co-mutations involving the second allele of RUNX1, or BCOR, PHF6, K-RAS, WT1 or TET2. Although curative in some patients, allogeneic stem cell transplantation from matched, un-related donors carries the risk of GVHD and relapse in FPD-MM. Thus, there is a need to develop novel targeted therapies to revert FPD-MM back to FPD. LINCS1000-CMap analysis, conducted with the RNA-Seq signature induced by lethal RUNX1 knockdown in AML cells with mutant (mt) RUNX1, was reported by us to reveal homoharringtonine (HHT or omacetaxine) and the anthelmintic fenbendazole (analog of mebendazole) as the top expression mimickers (EMs). In present studies, we demonstrate that treatment with HHT or mebendazole (MB) dose-dependently induced significantly greater loss of viability in six patient-derived (PD) samples of FPD-MM vs RUNX1-FPD or normal CD34+ progenitor cells. ATAC-Seq and H3K27Ac ChIP-Seq analyses conducted on BMA cells harvested longitudinally from the same patient at the RUNX1-FPD versus FPD-MM stage demonstrated greater ATAC-Seq peaks and chromatin accessibility in FPD-MM cells with increased mRNA expressions of MYB, MECOM and BCL2. Following HHT treatment, scRNA-Seq analysis demonstrated a marked reduction in the population of HSCs but concomitant increase in the macrophage population in FPD-MM (Fig. 1A). This was associated with positive enrichment of the mRNA expression belonging to the gene-sets (HALLMARK) of apoptosis signaling and TP53 targets while negatively enriching those of c-Myc targets. CyTOF analysis demonstrated that HHT treatment reduced c-Myc, EVI1, MCL1, BFL1 and CDK6 expression in phenotypically characterized FPD-MM stem-progenitor cells. Utilizing an FPD-MM sample from a patient with Runx1 K194N, we established the first ever cell line (GMR-AML1) expressing germline mtRUNX1. GMR-AML1 cells exhibited high surface expression of CD117 (c-KIT), CD123 (IL-3R) and CD33. CRISPR knockout (KO) RUNX1 led to growth inhibition, such that GMR-AML1 cells that retain Runx1 outcompeted Runx1 KO cells within 20 days. Treatment with HHT induced loss of viability in GMR-AML1 cells (LD50: 40 nM). HHT also caused concordant decline in ATAC-Seq and RNA-Seq peaks in ribosomal genes involved in protein translation, DNA POLR2B, RRM2, CCND2 and PTPN11. Treatment with MB also dose-dependently induced loss of viability of GMR-AML1 cells (LD50: 330 nM) as well as inhibited cell cycle and protein translation (via OPP Assay). CyTOF analysis revealed that MB treatment also depleted CDK6, RUNX1, c-Myc and EVI1 in phenotypically defined FPD-MM stem cells. Finally, in the tail-vein infused and engrafted, luciferized GMR-AML1 cell xenograft, compared to the vehicle control, treatment with omacetaxine or MB significantly reduced AML burden and improved overall survival of the NSG mice (p &amp;lt; 0.05) (Fig. 1B). These preclinical findings highlight the molecular features associated with progression of RUNX1-FPD to FPD-MM, including the newly established GMR-AML1 cell line. They also demonstrate that HHT or MB are effective against cellular models of FPD-MM versus RUNX1-FPD.

Multiomic Characterization of Myelodysplastic Neoplasms (MDS) with Micromegakaryocytes Highlights the Role of EZH2-RUNX1 deregulation in Disease Physiopathology and Response to Targeted Therapies

Micromegakaryocytes (micro-MKs), an unequivocal myeloid dysplastic feature in adults, are present in 15-20% of myelodysplastic neoplasms (MDS). We recently associated the detection of these small megakaryocytes with a mono- or bi-lobated nucleus in bone marrow (BM) smears of MDS patients with an unfavorable prognosis. The aim of the present study was to elucidate the molecular bases of micro-MK formation and its impact on MDS response to targeted therapies. We selected retrospectively a cohort of 40 MDS patient samples subjected to NGS myeloid panel and RNA sequencing and carried out a morphological re-evaluation that included micro-MKs recounting. Cases with isolated del(5q) or SF3B1 mutation were excluded. MDS cases were selected according to the following morphological findings: no dismegakaryopoiesis, dismegakaryopoiesis without micro-MK, low rate of micro-MK (&amp;lt;10%; micro-MK low) and high rate of micro-MK (&amp;gt;40%; micro-MK high). No patient harbored an intermediate (10-40%) rate of micro-MK. A comparative characterization of 8 representative patients was carried out subjecting diagnosis BM samples to Tapestri and Chromium single cell proteogenomic and transcriptomic platforms, gene set enrichment analysis, and deep cytogenetic characterization by optical genome mapping. Functional validation studies were performed using the megakaryoblastic cell line MEG01 differentiated to a mature MK phenotype by exposure to phorbol myristate acetate (PMA), as well as using the CRISPR-Cas9 tool for gene editing. Finally, activity of investigational drugs including 5-azacytidine, lenalidomide, venetoclax, rigosertib and pevonedistat, was evaluated in MEG01-activated cells and in vivo using a chicken embryo chorioallantoic membrane (CAM) model of MDS patient-derived xenograft (PDX). The most frequently mutated genes across all cohorts were ASXL1 (58%), SRSF2 (38%), RUNX1 (30%), TET2 (30%), STAG2 (20%), EZH2 (18%), DDX41 (10%), U2AF1 (10%), and ZRSR2 (10%) . Interestingly, EZH2 and RUNX1 mutations were enriched in the micro-MK high group ( EZH2: 0%, 10%, 14.3% vs. 66.7%, p 0.006; RUNX1: 0%, 20%, 35.7% vs. 83.3%, p 0.003), and both mutations co-occurred in 85.7% (6/7) of the EZH2 mutated cases. Single cell analyses revealed an enrichment of RUNX1 alterations in three populations within micro-MK high BM samples, including hematopoietic stem and progenitor cells, non-classical monocytes and immature erythroid cells. These samples were also characterized by a 5-fold increase in MK progenitor abundance and by the upregulation of a set of 10 EZH2-repressed genes in neutrophil-like cells and myeloid progenitors, with 7q loss presumably affecting the EZH2 locus in 50% of the cases with micro-MK. Using the in vitro MK differentiation model, we further demonstrated that EZH2 downregulation hampered the acquisition of MK surface markers CD41 and CD61, and that venetoclax was the sole agent retaining notable cytotoxic activity in differentiated cells when compared to undifferentiated megakaryoblasts. Accordingly, in a first-in-kind in vivo CAM-MDS transplant model, venetoclax was able to impair BM infiltration by human cells in a PDX model of micro-MK+ MDS. In summary, supporting the notion that EZH2 and RUNX1 exert a crucial role in the regulation of megakaryopoiesis, our present findings suggest that deregulation of EZH2-RUNX1 activity by loss-of-function mutation and/or genetic deletion affecting myeloid precursors, impairs a proper megakaryocytic differentiation process in MDS. Thus, this phenomenon may be considered as a characteristic feature of MDS cases with an elevated proportion of micro-MK. Of note, preliminary in vitro and in vivo results support the use of venetoclax for the treatment of this subgroup of patients with dismal outcome.