Lasho, Terra L.

6513 Background: PTPN11 (Ch12q24) is a proto-oncogene that codes for SHP2, a regulatory protein tyrosine phosphatase that impacts RAS/MAPK signaling. Germline variants have been described in juvenile myelomonocytic leukemia, while somatic variants have been associated with inferior survival in acute myeloid leukemia. PTPN11 variants are infrequent in chronic myelomonocytic leukemia (CMML), and their impact on disease phenotype and survival remains unclear. Methods: After IRB approval, 396 molecularly annotated CMML patients at Mayo Clinic were screened for somatic PTPN11 variants within 12 months of diagnosis while still in chronic phase disease. Overall (OS), acute myeloid leukemia (AML)-free survival (LFS), and predictors of survival were estimated using standard statistical measures. Results: 396 patients (median age 71 years; males 67%) with CMML were considered; pathogenic PTPN11 variants were seen in 12 (3%) with a median VAF of 20.5% (range: 4-50): A72T (n = 2), F285S (n = 2), and A72S (n = 2). Variants were most likely to be seen in the N-terminal SH2 domain (n = 7), followed by the PTP domain (n = 6), and the C-terminal SH2 domain (n = 1). PTPN11 variants clustered with CMML-2 (p = 0.02), higher absolute neutrophil count (p = 0.04), peripheral blood (p = 0.06) and bone marrow (p = 0.02) blast %, older age (p <0.01), and were less likely to have SRSF2 comutations (p = 0.02). After a median follow-up of 18 months (range 0-193), 253 (64%) deaths and 68 (17%) AML transformations were recorded. PTPN11 variants negatively and independently impacted both OS (median 13 vs. 31 months; p <0.01) and LFS (AML events: 42% (5/12) vs. 16% (63/384); p <0.01). In age-adjusted multivariate analysis restricted to genetic risk factors, survival was negatively affected by abnormal karyotype and mutations involving PTPN11, DNMT3A, and SETBP1, and positively by TET2 mutations. All but SETBP1 retained significance in the presence of additional independent risk factors, namely male sex (p <0.01) and leukocyte count (p <0.01); HR in the latter analysis was 4.3 for PTPN11, 2.8 for DNMT3A, and 1.6 for abnormal karyotype. A similar analysis for LFS identified, as risk factors, PTPN11 (p = 0.01), DNMT3A (p <0.01), and ASXL1 (p = 0.03) mutations, as well as bone marrow blast % (p <0.01), absolute monocyte count ≥10 (p = 0.02), and immature circulating myeloid cells (p <0.01). Conclusions: The current study identifies PTPN11 and DNMT3A mutations as independent risk factors for both OS and LFS in CMML. In regard to additional genetic risk factors, OS was positively affected by TET2 mutations and negatively by abnormal karyotype, while LFS was negatively affected by ASXL1 mutations.

Abstract Heterozygous germline variants in DDX41 are identified in ~5% of patients with Myelodysplastic Syndrome (MDS) or Acute Myeloid Leukemia (AML). Recognition of the hereditary nature of such cancers and a better understanding of the factors that affect progression to malignancy has deep implications for health surveillance of patients and relatives that share a pathogenic DNA variant. DDX41 has a role in ribosome biogenesis and in opposing R-loop accumulation at promoters, but the mechanisms through which DDX41 loss drives cancer development are poorly understood. In a collaboration between Cambridge, UK and Mayo Clinic, USA we identified 85 patients with germline DDX41 variants [73/85 with germline pathogenic variants (GPV) and 12 with variants of uncertain significance (VUS)]. The majority were identified through targeted myeloid NGS upon presentation with AML (30/85) or MDS (44/85). Average age at diagnosis was 65 (35-93) with male predominance (57/85). The most common GPV was the NM_016222.4(DDX41):c.415_418dup; p.D140Gfs*2 (15/85). The majority (53.4% 39/73), also acquired a somatic variants in the other DDX41 allele (referred to as GPV+Som). The most common somatic variant (82% 32/39) was the R525H, a variant predicted to reduce the ATPase function of the helicase domain. We hypothesise that the markedly reduced DDX41 activity in cells with biallelic variants, is a key cause of progression to overt malignancy. In our cohort, we observed that somatic variants occurred at a similar frequency in association with start loss, truncating, splice, in-frame deletion, and missense GPV. However, whereas MDS was the predominant diagnosis within GPV (67.6% 23/34, AML 26.4% 9/34), AML was the most frequent diagnosis within GPV+Som (AML 51%.2 20/39, MDS 38.4% 15/39). Overall, there was a significantly higher likelihood of AML in the presence of a somatic variant (p&lt;0.05), suggesting that there might be a dosage effect on the phenotype. This pattern was observed across all the GPV, with the exception of missense, suggesting that many might be benign polymorphysms. In addition, although somatic variants are typically subclonal (overall mean variant allele fraction VAF=9.87%, 3-27.8%), we observed a higher somatic VAF in AML over MDS cases (12.4% vs 7.7% respectively p&lt;0.05). A lower bone marrow cellularity was also characteristic of GPV+Som vs GPV alone (mean observed/expected cellularity for age 0.9 vs 1.32 respectively p&lt;0.05). In two cases, serial bone marrow morphology and NGS data were available, and we observed marked reduction in bone marrow cellularity upon acquisition of the R525H clone. The mechanisms of AML evolution appear different in DDX41 HMMS compared to sporadic cases. Interestingly, over half of our patients (21/34 GPV and 23/39 GPV+Som) did not exhibit additional somatic drivers. Our work suggests that acquisition of a somatic variant is a critical event in disease progression. Furthermore, the near universally subclonal nature of somatic variants suggest that they may affect disease phenotype through non-cell autonomous mechanisms. Citation Format: Ludovica Marando, Talha Badar, Yael Kusne, Audrey Morris, Terra Lasho, Abhishek Mangaonkar, Christy Finke, Carles Crawley, James M. Foran, Aref Al-Kali, Andrew King, Hassan Alkhateeb, Naseema Gangat, Chi Wong, Rong He, David Viswanatha, Faisal Basheer, Mark Litzow, Alejandro Ferrer, George Vassiliou, Mrinal Patnaik. Investigating the role of the somatic DDX41 variant in the context of DDX41 hereditary myeloid malignancy syndromes (HMMS) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A037.

Introduction: Telomere biology disorders (TBD) are multisystem diseases often associated with bone marrow failure (BMF) and pulmonary fibrosis. TBD are clinically diagnosed when mean telomere lengths (TL) in blood cells measured by clinical flowFISH assays fall &lt;1 st percentile, in comparison to age-matched reference controls. This information has limitations, e.g. TL in blood cells may not represent TL in other organs (e.g. lung, liver) and moreover, individuals with high clinical suspicion of TBD can show borderline average TL which complicates their diagnosis. The likely reason for this is that cell senescence can be triggered by shortening of a subset, or even a single chromosome arm telomere, a situation that may be missed when measuring average TL. Previous approaches to measure individual chromosome-arm telomere length (CA-TL) are only available for a small number of chromosome arms or can only estimate the number of shortened telomeres without matching them to specific chromosome arms. Taking advantage of recent developments in long-read next generation sequencing (NGS) and the availability of the recently published telomere-to-telomere (T2T) reference human genome, our group developed Telogator, (Stephens Z. et al, 2022) a bioinformatic tool that estimates CA-TL for most chromosome arms using long-read NGS data. Here we use Telogator to describe for the first time the CA-TL pattern in TBD patients in comparison with a control non-TBD cohort. Methods: TBD patients were identified through the Pre-Myeloid and Bone Marrow Failure Specialty Clinic at Mayo Clinic. All patients underwent flowFISH and genetic testing. Age-matched non-TBD patients identified at the Center for Individualized Medicine (Mayo Clinic) were used as reference controls. Informed consent was obtained from all individuals with approval from the Mayo Clinic Institutional Review Board. DNA from peripheral blood mononuclear cells was sequenced using PacBio Sequel II following the manufacturer indications for HiFi sequencing. BAM files were aligned to the T2T reference and CA-TL was calculated using Telogator as described in Stephens Z. et al. 2022. Average TL, CA-TL, and percent of short telomeres (both defined as &lt;5 kb and &lt;3.2 kb) were calculated. Comparisons of average TL and percent of short telomeres were performed using t-test, while Wilcoxon rank-sum test was used to compare CA-TL. Results: Four TBD patients [median age = 58.5 (52-60), 50% female] and 6 non-TBD controls [Median age = 56 (50-63), 33% female] were included in the analysis. All TBD patients presented average TL below the 1 st centile in both lymphocytes and granulocytes, and TBD-related phenotype (100% with pulmonary fibrosis, 50% with BMF, 25% with liver fibrosis). Three had a pathogenic variant in a TBD related genes [2 in TERT (NM_198253.3: c.2030G&gt;A, p.(Gly677Asp) and c.2515_2533del, p.(Thr839Alafs*29)) and 1 in NHP2 (NM_017838.3: c.459_460delAT; p.(X154Argext29))]. The average TL calculated by Telogator showed high correlation with flowFISH values (r 2 = 0.9883, p = 0.0059) and was lower in TBD patients than controls (p = 0.0012). Next, when comparing individual CA-TL between groups, only half of CA-TL was statistically different in the TBD group compared to controls (1p, 3q, 4p, 5p, 5q, 6q, 7p, 7q, 8q, 9p, 9q, 10q, 11p, 12q, 12q, 16p, 16q, 18q, 19p, 19q, 20p, 20q, 21q - Figure 1). The percentage of telomeres &lt;5kb and &lt;3 kb was significantly increased in TBD patients compared to controls (p= 0.014 and 0.0333, respectively). Discussion: Using a novel informatic tool, we replicate previous results obtained using classic approaches and were able to expand on these studies by measuring specific CA-TL in TBD patients. We identified statistically significant differences at the CA-TL level in these patients when compared to age-matched controls, while other CA-TL were not statistically different. Future studies and the inclusion of additional TBD patients will help define the clinical relevance of the differential CA-TL shortening and its prevalence among TBD patients. While limited by the small number of samples in the study, we show the feasibility of using this approach to study TL with unprecedented resolution and show that Telogator offers important advantages compared to classic methods to study telomere biology that can be used in other relevant fields like aging and/or cancer. Our results pave the way to advance the mechanistic understanding of TBD.

Germline variants in DDX41 are the most common cause of inherited predisposition to Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). DDX41 codes for an RNA helicase with many described functions, and thus the precise role of these variants in disease is uncertain. Approximately 2/3 of disease-associated DDX41 variants cause truncation of the DDX41 protein due to frameshift, loss of a translation start, or splice alterations, while the other 1/3 of DDX41 variants are missense. The effect of these missense variants on DDX41 function is largely unknown and thus they are typically categorized as variants of unknown significance (VUSs). Importantly, 30-50% of MDS/AML patients with either truncating or missense DDX41 variants acquire a somatic mutation in the other allele of DDX41, and this mutation has strong preference for the amino acid substitution R525H. Our previous work demonstrated that the R525H mutation causes loss of DDX41 function. To better understand how germline variants affect DDX41 function, we designed a genetic complementation strategy in immortalized hematopoietic progenitor cells with inducible deletion of DDX41. We screened six of the most common missense VUSs by expressing them from a lentivirus in DDX41-proficient cells and then inducing deletion of endogenous DDX41 to determine the effect of each VUS on proliferation and survival of the cells. Empty vector-transduced DDX41-deleted cells undergo cell-cycle arrest and apoptosis within 3-4 days. In contrast, viral expression of wild-type DDX41 rescues the survival and proliferation of the cells, while expression of the R525H mutant fails to rescue the cells. We screened the Y33H, M155H, G173R, R219H, P258L, and I396T variants of DDX41 in this assay, and all six of the mutants successfully rescued the survival and proliferation of DDX41-deleted cells long-term. This indicates that the mutant proteins coded for by these variants are functional. To test the effect of commonly observed truncating variants in the same assay, we expressed M1I, Q52fs, and D140Gfs variants of DDX41. Surprisingly, we saw complete rescue of cell proliferation and survival by the M1I and Q52fs variants. In contrast, the D140Gfs variant failed to rescue the cells. These data indicate that frameshift at D140 or later causes loss of DDX41 function. It is likely that translation from alternative methionine residues located at M127 or M132 of DDX41 accounts for the rescue of the cells by truncating variants affecting sites upstream of M127. In support of this theory, we targeted DDX41 for CRISPR-mediated knockout in MOLM13 cells and found that guide RNAs targeting exons 1-4, which are upstream of M127, produced homozygous frameshift mutations in proliferating cells from single-cell clones. However, guides targeting exon 6, which is downstream of M127 and M132, did not produce any surviving cells with homozygous frameshift mutations, despite screening 96 clones. This finding indicates that short DDX41 isoforms expressed from alternative start sites are functional and can support cell proliferation when full-length protein is lost. Collectively, these data raise important questions about the precise role of DDX41 variants in hematologic malignancy. Data from recently published patient cohorts indicates that patients with missense DDX41 variants have similar patient characteristics to those with truncating variants and thus these aberrations are thought to cause disease by similar mechanisms. While these cell line models may not fully recapitulate the pathogenic mechanism by which DDX41 variants cause MDS/AML predisposition, the results of this study demonstrate that the germline variants are not complete loss-of-function alleles in all cases and more complex mechanisms may be involved.

Introduction Chronic myelomonocytic leukemia (CMML) can be classified into dysplastic (dCMML) and proliferative (pCMML) subtypes based on a white blood cell count (WBC) ≥ 13 x 10 9/L for pCMML. Though this cutoff may seem arbitrary, there is evidence that pCMML is a unique biological entity with a distinct molecular profile and worse outcomes (Carr RM, et al. Nat Commun. 2021). We hypothesized that contemporary CMML prognostic scoring systems - such as the Mayo Molecular Model (MMM; Patnaik MM, et al. Leukemia. 2014) and the CPSS-Molecular Model (CMM; Elena C, et al. Blood. 2016) - would not adequately risk-stratify pCMML patients. Moreover, while truncating ASXL1 mutations are considered a high-risk feature in CMML, their prognostic relevance in pCMML is unknown. Methods After IRB approval, we compiled a large (n = 888), molecularly-annotated database of CMML patients seen at two US medical centers (Mayo Clinic and MD Anderson) via retrospective chart review. All statistical analyses considered the clinical and laboratory parameters obtained at the time of presentation. The cohort was divided into dCMML and pCMML subgroups, which were analyzed independently alongside the entire cohort. Categorical variables were compared by Fisher exact or Pearson χ 2 tests and continuous variables by Mann-Whitney U tests. Univariate and multivariate analyses were performed using Cox proportional hazards regression models. Survival was assessed via the Kaplan-Meier method. P-values &lt; 0.05 were considered significant. Calculations were performed using the BlueSky Statistics (v10.3.1) interface for R. Results We identified 474 (54%) dCMML cases and 410 (46%) pCMML cases with median age 71 years and 67% males. Patients with pCMML were more likely to have higher AMC (5.1 vs 1.5 x 10 9/L, p &lt; 0.0001), circulating immature myeloid cells (IMC; 65% vs 36%, p &lt; 0.0001) and abnormal karyotypes (37% vs 30%, p = 0.0187). They were also overrepresented in higher-risk categories of the MMM and CMM compared to dCMML (p &lt; 0.0001 for each model). The number of somatic mutations was higher in pCMML than dCMML (mean 3.0 vs 2.6, p = 0.0050). Mutations in ASXL1 (53% vs 39%), NRAS (21% vs 11%), CBL (18% vs 12%), SETBP1 (14% vs 7%), JAK2 (10% vs 3%), CEBPA (5% vs 2%), GATA2 (3% vs 0%), and FLT3 (3% vs 1%) were more frequent while mutations in TET2 (38% vs 48%), SF3B1 (2% vs 8%), ZRSR2 (2% vs 8%), and NPM1 (1% vs 3%) were less frequent in pCMML compared to dCMML, respectively (p &lt; 0.05 for all). Though the MMM and CMM effectively stratified the dCMML group, they did not provide meaningful risk stratification in the pCMML group (MMM p &lt; 0.05 overall but could not distinguish between low and intermediate-1/2 or intermediate-1 and high risks; CMM p &gt; 0.05). Likewise, there was no difference in the median overall survival (OS) or leukemia free survival (LFS) when pCMML patients were stratified by truncating ASXL1 mutations( Figure 1A). We then sought to better risk stratify pCMML. Univariate analysis identified increasing age, male gender, abnormal karyotype, and elevated WBC, ANC, and AMC as adverse predictors for OS. In addition, the presence of IMC and peripheral blood (PB) blast % were adverse factors for LFS, while age was not significant for LFS. None of the aforementioned mutations were significant in the univariate model for OS, whereas GATA2 and NPM1 mutations were significant for LFS. In multivariate analysis for OS, only male gender and AMC ≥ 10 x 10 9/L retained significance. In multivariate analysis for LFS, male gender, AMC ≥ 10 x 10 9/L, and peripheral blasts ≥ 5% remained significant; GATA2 and NPM1 mutations were not significant. These variables risk-stratified pCMML patients based on the presence of 0, 1, or ≥ 2 risk factors ( Figure 1B). In comparison, OS was 40.7 months (95% CI 35.2 - 50.1 mo.) for dCMML patients, 20.1 months (95% CI 18.2 - 22.7 mo.) for pCMML patients, and 31.8 months (95% CI 28.4 - 33.5 mo.) for the entire cohort. At last follow up, 586 deaths (66%) and 168 AML transformations (19%) were recorded in the cohort. Deaths in dCMML vs pCMML were 294 (62%) vs 291 (71%; p = 0.0067), with AML transformations in 83 (18%) vs 84 (21%; p = 0.2055) patients, respectively. Conclusions Truncating ASXL1 mutations, along with ASXL1-mutation weighted CMML prognostic models failed to effectively risk stratify pCMML patients. In pCMML, male gender and AMC ≥ 10 x 10 9/L, effectively risk stratified for OS, while these factors along with PB blasts ≥ 5% effectively risk stratified for LFS.

Background: Clonal cytopenia(s) of undetermined significance ( CCUS) is defined as persistent cytopenias arising in the context of myeloid neoplasm ( MN)-associated somatic mutations ( MT) in hematopoietic stem cells. Patients ( PTs) with TET2MT CCUS have a high probability of progression to MN. To date, no FDA-approved therapies exist for CCUS, and PTs often have similar cytopenias/transfusion needs as those with myelodysplastic syndromes ( MDS). TET2 is an ascorbic acid (AA)-dependent dioxygenase that catalyzes the conversion of 5-methylcytosine ( 5mC) to 5-hydroxymethylcytosine ( 5hmC), providing rationale to study high-dose IV (HI)-AA in TET2MT CCUS. Here we report the final results of this pilot trial. Methods: This is an investigator-initiated, single-institutional, phase II trial assessing the safety and efficacy of HI-AA in PTs with TET2MT CCUS (NCT03418038). PTs≥ 18 years with ≥1 TET2MT with or without additional somatic MTs, without prior treatment, and with any of the following laboratory criteria: (1) hemoglobin ( Hb) ≤10g/dL, (2) absolute neutrophil count ( ANC) ≤1(10^9/L), (3) platelet count ( PLT)≤100 (10^9/L) were eligible. HI-AA (1g/kg, maximum 100g) was given 3 times weekly for 12 weeks. The primary endpoint was hematologic response rates determined by MDS IWG 2018 criteria at week 20. (Platzbecker Blood 2018) and reported as hematologic improvement ( HI)- erythropoietic ( HI-E), platelets ( HI-P), neutrophils ( HI-N). Secondary endpoints include safety and adverse events ( AEs), graded by NCI-CTCAE v4.03. Correlative studies include changes in TET2MT variant allele fraction ( VAF), in vitro colony formation and differentiation, global and sequence-specific DNA methylation/hydroxymethylation, and quantitative 5hmC estimated by IHC in bone marrow biopsy specimens. Results: Ten patients were enrolled, with a median age of 71.4 (range: 65, 79) years, 8 (80%) males. The median number of mutations was 3, with 9 (90%) having co-mutations. All PTs but one [-Y] had normal karyotype. Baseline Hb levels were normal in all but one who was red blood transfusion dependent. Five (50%) PTs were thrombocytopenic, and 5 (50%) PTs had ANC&lt;1 (10^9/L). ( Table). HI-AA was well tolerated with the most common AEs being infusion-related polyuria (40%) and polydipsia (40%, 3 Grade 1, 1 Grade 2). One (10%) PT had constipation and headaches (both Grade 1), and 1 (10%) experienced dyspepsia. No treatment-related Grade 3 or 4 AEs' or deaths were reported. The median follow-up duration was 16.8 months (range: 9.4, 24.1). Overall, no PTs met the criteria for HI. There were no significant differences with regards to median Hb, PLT, and ANC values at baseline, week 20, and 1-year (median Hb: 13.3, 12.5, and 12.7 g/dL, p=0.93; PLT: 97 vs. 120 vs. 92.5X10^9/L, p&gt;0.99, and ANC: 0.9 vs. 1.2 vs. 1.3X10^9/L, p=0.99). By CTCAE grading criteria for cytopenias, 3 (30%) PTs had an improvement in severity of cytopenias, while 2 had worsening. One (10%) PT had an improvement in PLT (baseline vs. week 20: 62 vs 192X10^9/L) but was morphologically deemed to meet criteria for CMML-1 at week 20. Four (40%) PTs met criteria for disease progression (PT_4, 7,8,10); CMML-1- 2 and MDS-2. The median time to progression was 5 months (95% CI: 4.9, unreached). Two PTs had mutational clonal evolution, with 1 (PT_8) acquiring a RUNX1 (VAF: 20%) and 1 (PT_9) acquiring 2 CBL mutations (both VAF:4%) at one-year follow-up. There were no significant differences regarding the clinical characteristics or the TET2 variants between PTs who had stable disease vs. progression. There were no significant differences in TET2 VAF at baseline, week 20, and 1-year (median: 39% vs. 43% vs. 42%, p=0.54). We profiled changes in 5-mC and 5-hmC (Ilumina EpicArray) in PTs pre- and post-IV AA treatment and did not observe global changes. However, we were able to highlight site-specific changes in differentially methylated regions, largely resulting in hypomethylation at enhancer sites and affecting actively transcribed states in PTs with an improvement in cytopenia(s) severity by CTACE criteria ( Figure). Conclusion: We report the final safety and efficacy analysis of HI-AA in PTs with TET2 MT CCUS. The trial data suggest that HI-AA is safe and well tolerated. Although there were no significant responses by IWG MDS criteria, alleviation in severity of cytopenias in a subset of PTs (n=3) along with correlative epigenetic changes in enhancer regions, needs further exploration.

Introduction: Chronic myelomonocytic leukemia (CMML) is a myeloid neoplasm with limited therapeutic options and dismal outcomes. Truncating mutations in the epigenetic modifier ASXL1 are common and lead to a proliferative disease phenotype, resistance to epigenetic therapies, and higher rates of AML transformation. ASXL1-mutant CMML is characterized by widespread epigenetic remodeling and up-regulation of key leukemogenic driver genes (e.g. HOXA9 and its co-factor MEIS1). There is a genotype-specific activation of enhancers in ASXL1-mutant CMML that is associated with up-regulation of these leukemogenic driver genes. Here, we test the hypothesis that disrupting the oncogenic interactions at these genotype-specific enhancers can be exploited for therapeutic benefit. Methods: We identified activated enhancers specific to ASXL1-mutant CMML as previously described ( Binder et al. Nat Commun 2022;13(1):1434). To disrupt the E2542605 enhancer, we created unique mapped single guide RNAs (confirmed by BLAT). We generated a ribonucleoprotein complex using the Alt-R CRISPR-Cas9 system (IDT Technologies, Coralville IA). We combined the RNP along with Electroporation Enhancer to 3x10^5 cells of the monocytic cell line U937 in 20uL strip-wells provided by the SF Cell Line 4D-Nucleofector X Kit (Lonza, Basel, Switzerland). To deliver the RNP, U937 cells were electroporated via listed cell line specific protocol by the Amaxa 4D-Nucleofector (Lonza). Forty-eight hours post-electroporation, cells were split and sequenced for E2542605 genomic structure disruption. In parallel, RNA was harvested, converted to cDNA and qPCR for HOXA9 and MEIS1 expression was performed using PrimeTime qPCR FAM labeled probe assays (IDT). Gene expression was determined using delta-delta Ct method. For assessment of selective sensitivity of ASXL1 / enhancer interactions, we performed progenitor colony forming assays using primary bone marrow mononuclear cells from CMML patients with or without ASXL1 mutations and healthy normal controls (n=2 in each category), exposed to increasing concentrations of the dual p300/CBP and BET bromodomain inhibitor EP31670. Briefly, we plated cells in methocellulose fortified with recombinant human cytokines supporting hematopoietic growth in duplicate, in a range of EP31670 concentrations. After 10 days, visible hematopoietic colonies were quantified and characterized. IC 50 values were generated using non-linear regression log(EP31670) vs. normalized response. Results: We computationally identified candidate transcriptional regulators enriched at the activated enhancers specific to ASXL1-mutant CMML, including BRD4 (FDR&lt;2.57x10 -370), CBP (FDR=2.56x10 -217), and p300 (FDR=6.36x10 -93). We selected the ASXL1-mutant-specific enhancer E2542605 (predicted to drive HOXA9 expression), given the up-regulation of HOXA9 in ASXL1-mutant CMML (log 2-fold change +2.05, FDR=0.006, n=14). We disrupted E2542605 using two different CRISPR guides, leading to a PCR trace detection of a 14bp deletion and a 26bp indel in the E2542605 enhancer region, respectively. Both enhancer disruptions led to a down-regulation of its putative target gene HOXA9: Mean relative fold-change 0.36 (p=0.001, n=3) and 0.65 (p=0.046, n=3), respectively ( Fig. 1a). We then used a pharmacologic approach (EP31670) to target the candidate transcriptional regulators involved in the oncogenic interaction at these ASXL1-mutant-specific enhancers. We observed selective sensitivity to growth of CMML colonies (particularly ASXL1-mutant colonies) compared to ASXL1-wild type cases and normal bone marrow controls ( Fig. 1b). Conclusions: In CMML patients, truncating ASXL1 mutations are associated with the activation of genotype-specific enhancers and the recruitment of an extended repertoire of transcriptional regulators including BRD4, CBP, and p300. In a model system, the disruption of an enhancer predicted to govern the expression of the leukemogenic driver HOXA9, led to a significant down-regulation of HOXA9 expression. Pharmacologically disrupting the oncogenic interaction at such enhancers with EP31670 in primary patient samples led to significant therapeutic effects at nanomolar drug concentrations in a genotype-specific manner. Oncogenic interactions at activated enhancers are viable therapeutic targets in ASXL1-mutant CMML and warrant further clinical investigation.

INTRODUCTION Telomere biology disorders (TBDs) are multisystem diseases caused by accelerated telomere shortening that can present with bone marrow failure and pulmonary fibrosis, among other abnormalities. TBD diagnosis is considered when average blood telomere length (TL) measured by clinical flowFISH is &lt;1 st percentile and/or a pathogenic variant in telomere maintenance genes (most commonly TERT, TERC, RTEL1) is identified. Pathogenic variants in genes coding for the telomerase protein ( TERT) and RNA component ( TERC) decrease telomerase activity. Similarly, variants in RTEL1 result in defective unwinding of the t-loop at telomere ends, increasing the levels of free t-circles in cells. Variants in these genes are often classified as variants of uncertain significance (VUSs by ACMG criteria) with additional efforts needed for variant curation. To address this, we describe results of functional testing performed in TBD patients with VUSs involving TERT, TERC, and RTEL1. METHODS TBD suspected patients were identified through the Pre-Myeloid Cancer and Bone Marrow Failure Clinic (Mayo Clinic) where they underwent clinical flowFISH and genetic testing for TBD related genes. Informed consent was obtained with approval from the Mayo Clinic Institutional Review Board. Viable peripheral blood and bone marrow samples were collected along with age matched healthy volunteers. Telomerase activity was measured in samples with TERT and TERC variants through telomeric repeat amplification protocol (TRAP) assays using the TRAPeze XL Telomerase Detection Kit (MilliporeSigma) following manufacturer instructions. HCT116 cells were used as positive controls while heat inactivated samples served as negative controls. Fluorescence was measured in a SpectraMax plate reader. Percentage of total product generated (TPG) relative to healthy age matched controls was calculated. T-circle detection assays were used on RTEL1 samples based on the protocol by Zhang et al. (2017). Final southern blotting was completed using the TeloTAGGG Telomere Length Assay Kit (Roche), and t-circle bands were visualized in a c600 GEL Imaging System (Azure Biosystems). RESULTS Ten patients (mean age = 53 years (27-71), 50% female) were included in our study. Patients 1 to 5 presented VUS in TERT (n=4) or TERC (n=1) and TL &lt;1 st percentile in lymphocytes and/or granulocytes. Patient 6 carried a known pathogenic variant in RTEL1 and was included as a positive control. Patients 7 to 9 with RTEL1 variants presented TL close to the 10 th or between the 1 st-10 th percentile. No TL results were available for patient 10 but was tested due to the presence of a VUS in RTEL1 (Table 1). Similarly, while patient 7 carried a likely pathogenic variant, it was tested due to presenting lymphocyte TL between the 1 st-10 th percentile. TRAP results indicated decreased telomerase activity compared to age matched controls in all samples with TERT and TERC variants suggesting a pathogenic effect (52.%, 10.7%, 48.7%, 4.4% and 5.2% of TPG compared to age matched controls, respectively) (Table 1). On the other hand, t-circles were only seen in patient 6 (Figure 2). DISCUSSION We used TRAP and t-circle assays to explore the telomerase activity and presence of t-circles in patient samples with TERT/TERC and RTEL1 variants. In the case of TERT/TERC, we observed a reduction of telomerase activity in all samples. Together with blood TL below the 1 st percentile, this suggests that these variants are indeed deleterious (Table 1). However, since patient 4 presented two TERT VUSs, additional testing is needed to conclude how each variant impacts telomerase activity independently. In regard to RTEL1 variants, we observed t-circles in patient 6 as expected. No t-circles could be seen on all remaining samples, including patient 7 who carried a variant classified as likely pathogenic (Figure 1). This in-frame deletion has been described once in a TBD family (Cogan et al. 2015) but was not functionally tested. This result, together with the borderline TL seen in this patient, may suggest that some compensatory mechanism exists on this sample, or that the deleterious effect of the variant is not strong enough to be detected by our assay. Further studies are currently ongoing by our group to validate these results using a cell-based transfection disease model. In summary, we show that functional testing can be a helpful tool to determine pathogenicity of VUSs in TERT/TERC and RTEL1.

Introduction: Clonal dendritic cell (DC) aggregates in chronic myelomonocytic leukemia (CMML) are seen in approximately 20-30% patients at diagnosis. Previously, these were thought to be comprised of CD123+ plasmacytoid DCs but our data has suggested that these are heterogenous and comprise of myeloid DCs, monocytes, and myeloid-derived suppressor cells. Furthermore, these aggregates uniformly express immune checkpoints such as indoleamine 2,3-dioxygenase-1 (IDO), associate with an IDO-specific systemic impact (increased trytophan catabolism), and expanded regulatory T cell population in CMML, suggesting a role in inducing immune tolerance. DC aggregates are also associated with increased risk of acute myeloid leukemia (AML) transformation and resistance to hypomethylating agents, highlighting that they are important for disease progression in CMML (Mangaonkar A et al. Blood Adv 2020). We performed this study to decipher the architecture of a CMML DC aggregate to understand the complex inter-cellular interactions, and assess their impact on the adaptive immune system. Methods: In this study, we used archival fresh-frozen paraffin-embedded (FFPE) bone marrow biopsy specimens obtained from CMML patients, and isolated monocytes (CD14+ magnetic bead sorting) and lymphocytes (CD14-ve fraction) from untreated CMML and age-matched normal control (NC) peripheral blood mononuclear cells (PBMCs). DCs were cultured from monocytes using a commercial protocol. Spatial architecture of the CMML BM microenvironment was interrogated through multiplex imaging of FFPE samples by co-detection by indexing (CODEX) using 30 markers (CCR6, CD107a, CD11c, CD20, CD21, CD31, CD3e, CD4, CD44, CD45, CD45RO CD56, CD68, CD8, E-Cadherin, FoxP3, HLA-DR, CD14, CD16, CD11b, CD33, CD15, CD66b, CD123, CD303, CD34, CD163 and IDO). Single-cell transcriptomic signatures were analyzed using the 10X single cell Fixed RNA profiling platform, and validated using bulk RNA seq (Illumina stranded mRNA prep) from isolated and cultured cells. Impact of DCs on lymphocytes was assessed via autologous mixed DC-T culture experiments. Results: BM FFPE samples from 5 CMML cases (3 with DC aggregates) were included for multiplex imaging via CODEX. Results confirm the significant interactions of CD8+ and CD4+ T cells with CD123+/HLA-DR+ DCs within a CMML DC aggregate (Fig. 1A). Upon quantitative assessment of cell-cell interactions, IDO+/CD8+T ( P=0.006), IDO+/MDSC ( P=0.005), and IDO+/macrophage ( P=0.001) interactions were significantly increased in CMML samples with versus without DC aggregates . Specific findings were validated using single-stain immunohistochemistry and multiplex immunoflourescence staining. Next, in order to assess transcriptomic signatures within BM at the single-cell level, we chose two cases; CMML (BM aspirate blast% 1, WBC 49.6 x 10 9/L, ASXL1/GATA2/SETBP1/SRSF2 mutations) and AML transformed from CMML (BM aspirate blast% 81, WBC 152 x 10 9/L, NPM1/DNMT3A/TET2 mutations). Approximately, 10,000 cells were obtained and analyzed with cell type annotation through human PBMC reference (Azimuth). Results show an expanded regulatory T cell population in AML compared to CMML (UMAP plot, Fig. 1B), suggestive of immune tolerance. In order to validate findings and perform functional experiments, we used sorted monocytes and cutured DCs from CMML and NC. DC phenotype was confirmed both through visual inspection and flow cytometry, and clonality was confirmed through Sanger sequencing (for previously identified mutations). Differential gene expression analysis through bulk RNAseq showed significant differences between CMML and NC lymphocytes with immunoinhibitory receptor, LILRB2 (log2fold change=7.05, adjusted P=0.0002) being one of the top five significantly upregulated genes, further confirming immune tolerance. Finally, mixed autologous DC-lymphocyte reaction (1:4 ratio) experiments demonstrated that DCs induce T cell tolerance in CMML (expansion of exhausted PD1/TIM3/LAG3+ CD8 T cells) when compared to NC. Conclusion: Clonal dendritic cell aggregates within the CMML bone marrow microenviroment interact with T cells and induce immune tolerance. This represents a novel mechanism of disease progression in CMML with potential therapeutic targets (IDO, LILRB2, and/or CD123).

Introduction HMAs are the only approved therapy for CMML. In order to improve response rates and survival, the addition of VEN has been utilized in both CMML and after blast transformation (CMML-BT) with conflicting results in small single center studies. To clarify the role of upfront HMA+VEN in CMML and CMML-BT, we performed a large multicenter analysis. Methods We included patients with HMA-naïve CMML and CMML-BT treated with combination HMA and VEN from 4 academic centers. The primary outcome was overall survival (OS) with secondary outcomes including overall response rate (ORR) by 2015 MDS/MPN IWG criteria for CMML and 2022 ELN criteria for CMML-BT. To better appreciate the benefit of adding VEN to HMA treatment, we also performed 1:1 propensity score matched (PSM) analysis of patients treated with HMA alone as upfront therapy for CMML or CMML-BT. Matching was based on age, sex, ECOG performance status, CPSS-Mol, blood counts, peripheral and bone marrow blasts. Results Eighty-nine patients were treated with frontline HMA+VEN, 51 CMML and 38 CMML-BT (5 had received HMA alone for prior CMML). The median age was 71 years, 70% were male, and 83% had ECOG 0-1. Among CMML patients, 55% had MP-CMML subtype, 65% had CMML-2 and 12%, 30%, 58% were CPSS-mol intermediate-1, intermediate-2 and high risk, respectively. In the entire cohort, ASXL1 (63%), TET2 (47%), SRSF2 (39%) and RUNX1 (27%) were the most commonly mutated genes and 55% of patients harbored a RAS mutation ( KRAS [20%], NRAS [18%], CBL [13%], NF1 [11%], PTPN11 [9%]). For HMA combination partner, 46 (52%) were treated with azacitidine, 35 (39%) with decitabine, and 8 (9%) with both during their VEN course. During a median follow-up time of 15.8 months (mos) 52 patients (58%) died: 23 (45%) with CMML and 29 (76%) with CMML-BT. Among CMML patients, the ORR was 90%, including complete remission (CR) in 22%, complete cytogenic remission (CCR) in 4%, optimal marrow response (CMR) in 50%, and partial marrow response (PMR) in 6%. The OS of CMML patients was 25.1 mo (95% CI 15.7 - NR). A PSM population of HMA patients matched to HMA+VEN treated patients showing similar baseline characteristics. ORR was significantly higher with HMA+VEN (95%) as compared those treated with HMA alone (46%), p &lt; 0.001. However, there was no significant OS difference between HMA+VEN vs HMA alone (19.1 mos vs 19.1 mos, p = 0.85, Figure 1). For CMML-BT patients, the ORR of HMA+VEN was 81% including CR in 17%, CR with partial hematologic recover (CRh) in 3%, CR with incomplete hematologic recovery (CRi) in 28%, morphologic leukemia-free state (MLFS) in 22% and partial response (PR) in 11%. The OS of CMML-BT patients was 8 mos (95% CI 4.6 - 26.2). A PSM population of CMML-BT patients treated with HMA alone showed a significantly increased ORR with HMA+VEN (82% vs 40%, p = 0.039). However, there was no difference in OS (7.49 mos vs 9.26 mos, p = 0.83, Figure 2). Among HMA-VEN treated patients, achievement of a CR was associated with an improved OS in the CMML (HR 0.31, 95% CI 0.13 - 0.75) and CMML-BT (HR 0.37, 95% CI 0.19 - 0.74). No predictors of response were identified, including the presence of a RAS mutation. In terms of safety, 7 patients (7.9%) had a grade 3/4 (G3/4) neutropenic infection and 2 patients (2.2%) had a G3/4 hemorrhagic event while being treated with HMA+VEN. A total of 29 patients went on to receive allogeneic transplant (alloSCT): 22 (43%) with CMML and 7 (18%) with CMML-BT. CMML patients who underwent alloSCT after HMA+VEN were younger as compared to non-transplanted patients (64 vs 74 years, p &lt; 0.0001), but had otherwise similar baseline characteristics including stage, CPSS-mol category and prevalence of RAS mutations. Similarly, CMML-BT patients bridged to alloSCT were younger (66 vs 74 years, p &lt; 0.002) and had a better ECOG performance status (p = 0.047). Of patients who received alloSCT, 10 patients (34%) died, 5 with CMML and 5 with CMML-BT. The median OS after alloSCT was 26.7 mo. Conclusions Although the use of VEN with HMA in the upfront treatment of CMML and AML secondary to CMML results in higher response rates compared to HMA alone, this combination does not improve survival based on this multi-center PSM analysis. The current sample size of CMML-BT may limit detection of OS differences between treatment groups. The role of VEN to reduce blasts and serve as a bridge to alloSCT, particularly after HMA failure, requires further investigation in larger studies in both the CMML and CMML-BT populations.