When to worry about inherited bone marrow failure and myeloid malignancy predisposition syndromes in the setting of a hypocellular marrow

With our increasing understanding of inherited marrow failure and myeloid malignancy predisposition syndromes, it has become clear that there is a wide phenotypic spectrum and that these diseases must be considered in the differential diagnosis of both children and adults with unexplained defects in hematopoiesis. Moreover, these conditions are not as rare as previously believed and may present as aplastic anemia, myelodysplastic syndrome, or malignancy over a range of ages. Establishing the correct diagnosis is essential because it has implications for treatment, medical management, cancer screening, and family planning. Our goal is to highlight insights into the pathophysiology of these diseases, review cryptic presentations of these syndromes, and provide useful references for the practicing hematologist.

Impaired myelopoiesis in congenital neutropenia: insights into clonal and malignant hematopoiesis

A common feature of both congenital and acquired forms of bone marrow failure is an increased risk of developing acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Indeed, the development of MDS or AML is now the major cause of mortality in patients with congenital neutropenia. Thus, there is a pressing clinical need to develop better strategies to prevent, diagnose early, and treat MDS/AML in patients with congenital neutropenia and other bone marrow failure syndromes. Here, we discuss recent data characterizing clonal hematopoiesis and progression to myeloid malignancy in congenital neutropenia, focusing on severe congenital neutropenia (SCN) and Shwachman-Diamond syndrome. We summarize recent studies showing excellent outcomes after allogenic hematopoietic stem cell transplantation for many (but not all) patients with congenital neutropenia, including patients with SCN with active myeloid malignancy who underwent transplantation. Finally, we discuss how these new data inform the current clinical management of patients with congenital neutropenia.

PLAGL2 Independently Drives Aberrant Erythropoiesis and Initiation of Preleukemic State

Background The identification and understanding of early drivers in malignancy is crucial to prevent or revert preleukemic events. Del(20q) is one of the most common primary cytogenetic abnormalities found in preleukemic malignancies from myeloproliferative neoplasms to myelodysplastic syndrome (MDS). Previous studies have identified a "common retained region" within 20q11.21 that is often amplified in a subset of MDS patients. PLAGL2 is one of the 4 genes identified to be within the minimally conserved amplified region. (MacKinnon et al. 2010) Indeed, in previously published datasets of MDS hematopoietic stem and progenitor cells (HSPCs) transcriptome, PLAGL2 is significantly elevated in del(20q) patients compared to healthy controls. However, we have found that its level is also higher in HSPCs of cytogenetically normal MDS patients with low blasts. Given these findings, we sought to define the role of PLAGL2 as a potential early driver of myeloid malignancies. Results In healthy cord blood (CB) HSPCs, PLAGL2 overexpression enhanced proliferation ex vivo, better maintained stemness and decreased apoptosis. Colony formation assays also identified increased output of the erythroid lineage. Xenotransplanted CB CD34+ HSPCs overexpressing PLAGL2 exhibited increased engraftment competitiveness and led to splenomegaly with signs of hypercellularity after 20 weeks, features consistent with clinical observations of hematological malignancy. Grafts derived from PLAGL2 overexpressing cells reproducibly maintained a significantly larger CD34+ HSPC compartment. Intriguingly we also identified that ~50% of PLAGL2-overexpressing grafts exhibited a significant erythroid (CD71+/CD235a+) component where none was observed in the control group. This unique finding of aberrant erythropoiesis is reminiscent of clinical observations in patients with 20q11.21 amplification, where a high proportion of erythroblasts in the marrow and in some cases progression to erythroleukemia was noted. To evaluate the progression of PLAGL2-overexpressing grafts, further secondary transplantations were carried out and showed the persistence of only immature erythroid progenitors (CD71+/CD235a-) coupled with a near complete absence of lymphopoiesis in the same grafts. Together, our data strongly suggests ectopic levels of PLAGL2 can independently drive the expansion of human HSPCs and enforce features of myeloid malignancy. To uncover the molecular mechanism underlying PLAGL2 function, we performed RNA-seq and Cut&Run in human CB CD34+ HSPCs overexpressing PLAGL2. Geneset enrichment analysis of the transcriptome and over-representation analysis of bound genes both identified signatures consistent with LSCs. We compared these findings with identically-derived omics profiles of HSPCs overexpressing PLAG1, a closely related family member that our lab has identified as a potent expander of HSCs ex vivo but not capable of promoting malignant features. We found a strong common feature in the downregulation of ribosomal components and translation machinery, then functionally validated reduced protein synthesis in PLAGL2 overexpressing HSPCs through OP-Puro assays. We have shown dampened mRNA translation to be one of the mechanisms by which PLAG1 enhances stemness and survival of HSCs, one that potentially extends to PLAGL2 as well. However, we also identified discordant signatures, notably PLAGL2's unique capacity to reduce mitochondrial translation, a pathway associated with ineffective erythropoiesis and MDS and one that we are currently exploring as a means by which PLAGL2 can enforce malignant phenotypes. Finally, to investigate the potential of PLAGL2 as a therapeutic target in MDS, we performed shRNA knockdown in MDSL, a human MDS cell line. In vitro competitive assays with mixed wildtype cells showed steady dropout of PLAGL2 depleted cells. Currently, we are continuing to purse the dependence of primary MDS cells on PLAGL2 through in vivo xenograft models. Conclusion We have identified PLAGL2's potential as an early independent driver of myeloid malignancy and aberrant erythroid differentiation. An understanding of PLAGL2 and its downstream mechanisms will not only further our understanding on the development of early myeloid malignancies but also potentially provide another avenue to treat or prevent leukemia before it manifests. Disclosures Dick: Celgene, Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Impact of the COVID-19 Pandemic on Primary Care Access for Patients with Hematologic Malignancies

Introduction: Primary care physicians are essential to cancer care. They frequently identify signs and symptoms leading to a diagnosis of cancer, and provide ongoing support and management of non-cancer health conditions during cancer treatment. Both primary care and cancer care have been greatly affected by the COVID-19 pandemic. In the United States, cancer-related patient encounters and cancer screening decreased over 40% and 80% respectively in January to April 2020 compared to 2019 (London et al. JCO Clin Cancer Inform 2020). However, the impact of the COVID-19 pandemic on primary care access for cancer patients remain unclear. Methods: We undertook a population-based, retrospective cohort study using healthcare databases held at ICES in Ontario, Canada. Patients with a new lymphoid or myeloid malignancy diagnosed within the year prior to the COVID-19 pandemic, between July 1, 2019 and September 30, 2019 (COVID-19 cohort) were compared to patients diagnosed in years unaffected by the COVID-19 pandemic, between July 1, 2018 - September 30, 2018 and July 1, 2017 - September 30, 2017 (pre-pandemic cohort). Both groups were followed for 12 months after initial cancer diagnosis. In the COVID-19 cohort, this allowed for at least 4 months of follow-up data occurring during the COVID-19 pandemic. The primary outcome was number of in-person and virtual visits with a primary care physician. Secondary outcomes of interest included number of in-person and virtual visits with a hematologist, number of visits to the emergency department (ED), and number of unplanned hospitalizations. Outcomes, reported as crude rates per 1000 person-months, were compared between the COVID-19 and pre-pandemic cohorts using Poisson regression modelling. Results: We identified 2882 individuals diagnosed with a new lymphoid or myeloid malignancy during the defined COVID-19 timeframe and compared them to 5997 individuals diagnosed during the defined pre-pandemic timeframe. The crude rate of in-person primary care visits per 1000 person-months significantly decreased from 574.4 [95% CI 568.5 - 580.4] in the pre-pandemic cohort to 402.5 [395.3 - 409.7] in the COVID-19 cohort (p < 0.0001). Telemedicine visits to primary care significantly increased from 5.3 [4.8 - 5.9] to 173.0 [168.4 - 177.8] (p < 0.0001). The rate of combined in-person and telemedicine visits to primary care did not change from 579.8 [573.8 - 585.8] in the pre-pandemic cohort to 575.5 [566.9 - 584.2] in the COVID-19 cohort (p = 0.43). In-person visits to hematologists decreased from 504.1 [498.5 - 509.7] to 432.8 [425.3 - 440.3] (p < 0.0001), and telemedicine visits to hematologists increased from 6.6 [6.0 - 7.3] to 75.9 [72.8 - 79.1] (p < 0.0001). The rate of combined visits to hematologists did not change from 510.7 [505.1 - 516.4] to 508.7 [500.6 - 516.8] (p = 0.68). The rate of ED visits significantly decreased from 95.1 [92.7 - 97.6] in the pre-pandemic cohort to 84.7 [81.4 - 88.0] in the COVID-19 cohort (p < 0.0001). The rate of unplanned hospitalizations did not change from 64.8 [62.8 - 66.8] to 65.7 [62.9 - 68.7] (p = 0.60). Conclusions: Primary care visits for patients with hematologic malignancies did not significantly change during the pandemic, but there was a sizeable shift from in-person to telemedicine visits. Similar findings were seen for visits to hematologists. While the rate of visits to the ED decreased, potentially due to concern of being exposed to the COVID-19 virus, the shift in ambulatory practices did not seem to impact the rate of unplanned hospitalizations. Disclosures No relevant conflicts of interest to declare.

Homoharringtonine and Mebendazole Are Preferentially Lethal Against Models of Myeloid Malignancy Associated with Germline Mutant RUNX1

RUNX1 is a master-transcriptional regulator involved in normal and malignant hematopoiesis. Majority of mono-allelic germline mutations in RUNX1 are missense, large deletions or truncation mutations, behaving mostly as loss of function (LOF) mutations. They are ~40%-penetrant and cause Familial Platelet Disorder (RUNX1-FPD) that has a propensity to evolve into myeloid malignancy (FPD-MM), i.e., MDS or AML. FPD-MM harbors co-mutations, most commonly on the second allele of RUNX1, and on BCOR, PHF6, K-RAS, WT1 or TET2, which confer relative resistance to standard therapy for MDS or AML. Although curative in some patients with FPD-MM, allogeneic transplantation from matched, un-related donors carries risk of graft versus host disease and frequent AML relapse. This creates a strong rationale and an unmet need to develop novel targeted therapies for FPD-MM. We previously reported on utilizing the RNA-Seq signature of RUNX1 knockdown, which exerted more lethality in AML cells with mutant (mt) RUNX1 compared to AML harboring two copies of wild-type RUNX1, for conducting LINCS1000-CMap analysis. This identified several expression mimickers (EMs), including the protein synthesis inhibitor homoharringtonine (HHT or omacetaxine) and anthelmintic fenbendazole (analog of mebendazole). Present studies demonstrate that treatment with HHT or mebendazole (MB) dose-dependently induced significantly greater loss of viability in four patient-derived (PD) bone marrow aspirate (BMA) samples of FPD-MM (3 AML and 1 MDS) compared to RUNX1-FPD (3 samples) or in normal CD34+ progenitor cells. In a patient with RUNX1-FPD (expressing mtRUNX1 K194N), who developed FPD-MM, following co-mutations were documented by NGS: BCOR A1437fs, PHF6 L324fs, SF3B1 D781G and SRSF2 P95R/L. From BMA of this patient, we successfully established the first ever, continuously cultured cell line (GMR-AML1) expressing the same germline mtRUNX1. GMR-AML1 cells were cytogenetically diploid and lacked MYC or MLL1 rearrangement, or any copy number gains or losses on array CGH. However whole exome sequencing (WES) identified additional mutations in TP53 (P72R), AIM2 (K340fs), NELFB (L523F), CEP152 (Y370X), SUGP2 (H23L), RRM2B (R71fs), TADA3 (T27R), SPDYE6 (G292C) and PRDM9 (S814R) with % VAF ranging between 33 to 55%. GMR-AML1 cells exhibited high surface expression of CD117 (c-KIT), CD123 (IL3R), CD86 and CD33, but without expression of CD34, CD14, CD11b, MPO or CD135 (FLT3). Compared to the AML OCI-AML5 cells with somatic mtRUNX1, GMR-AML1 cells demonstrated markedly reduced protein expression of RUNX1, RUNX2, PU.1, c-Myb, GFI1, GFI1B, FLT3, MEIS1 and CEBPα (p42), but much higher protein expression of RUNX3 and NOTCH (p120). CRISPR-Cas9 knockout of RUNX3 in GMR-AML1 cells restored RUNX1 expression, while significantly increasing % of differentiated cells. Although dose-dependently sensitive to daunorubicin, etoposide, cytarabine and panobinostat (class I and II HDAC inhibitor), GMR-AML1 cells were relatively insensitive to venetoclax, A1155463 (Bcl-xL inhibitor), AZD-5991 (MCL1 inhibitor), azacytidine or decitabine. Notably, treatment with HHT or MB dose-dependently induced loss of viability of GMR-AML1 cells (LD50: 40 and 330 nM, respectively). Additionally, co-treatment with HHT and venetoclax synergistically induced apoptosis in GMR-AML1 cells, as determined by the SynergyFinder algorithm. This synergy in GMR-AML1 cells was associated with abrogation of venetoclax-induced increase in MCL1 and Bcl-xL levels, as well as greater decline in levels of RUNX3, PU.1, c-Myb, c-Myc, MPL and CDK4/6. Tail vein infusion and engraftment of luciferase-transduced GMR-AML1 (10 6 cells) caused marked splenomegaly and 100% mortality of NSG mice by day-18, post-infusion. We will present at the ASH meeting findings of ongoing in vivo studies determining effects of treatment with HHT and/or venetoclax, versus vehicle control, on AML burden and overall survival of NSG mice engrafted with GMR-AML1 cells. Overall, preclinical findings presented here highlight the molecular and genetic features associated with progression of RUNX1-FPD to FPD-MM, especially in the newly established GMR-AML1 cell line. They also demonstrate that HHT or MB are preferentially more lethal against FPD-MM versus RUNX1-FPD cells and exert synergistic lethality with venetoclax against GMR-AML1 cells. Disclosures DiNardo: GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; AbbVie: Consultancy, Research Funding; Agios/Servier: Consultancy, Honoraria, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; Takeda: Honoraria; ImmuneOnc: Honoraria, Research Funding; Forma: Honoraria, Research Funding; Foghorn: Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. Takahashi: GSK: Consultancy; Celgene/BMS: Consultancy; Symbio Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy. Khoury: Stemline Therapeutics: Research Funding; Angle: Research Funding; Kiromic: Research Funding.

Inherited Blood Cancer Predisposition through Altered Transcription Elongation

Despite considerable advances in defining the somatic driver mutations underlying myeloid malignancies, including the myeloproliferative neoplasms (MPNs), a significant heritable component for these diseases remains poorly understood. While common genetic variant association studies have been valuable, they fail to explain the majority of heritable variation. We reasoned that rare variant association studies could provide a valuable complementary approach to identify additional inherited risk factors. We therefore utilized exome sequencing data from 166,953 UK Biobank participants and performed a gene-based burden analysis for germline genetic variants conferring risk for acquiring a myeloid malignancy. CTR9, which encodes a key component of the PAF1 transcription elongation complex, was among the significant genes identified (SKAT-O p-value = 5.47x10 -7). The deleterious variants in CTR9 collectively exhibit a 9.6 (95%CI = 4.86-19.04) increased odds of acquiring a myeloid malignancy and this risk was largely driven by the MPNs. We replicated this association in an independent cohort of 211 MPN patients using external controls. We could show through structural and biochemical analyses that the identified deleterious variants perturbed assembly of the PAF1 complex but did not display dominant negative activity. Given that increased hematopoietic stem cell (HSC) self-renewal has been shown to predispose to the risk of acquiring MPNs, we sought to define whether CTR9 perturbation could alter HSC self-renewal or function. We achieved predominantly heterozygous loss-of-function in human hematopoietic stem and progenitor cells (HSPCs) by titrating Cas9 ribonucleoprotein delivery with several independent guide RNAs. Partial loss of CTR9 in HSPCs resulted in expansion of phenotypic long-term HSCs (LT-HSCs) and more differentiated short-term HSCs (ST-HSCs). We additionally could show through single cell RNA-sequencing (scRNA-seq) that there was an expansion of molecularly defined HSCs upon partial loss of CTR9. The observed increase in HSCs appeared paradoxical, given that the PAF1 complex has been suggested to be crucial for HSC maintenance. To explore how the observed HSC expansion with CTR9 perturbation may arise, as well as given known interactions between the PAF1 complex and the competing transcriptional super elongation complex (SEC), we examined whether SEC target genes in HSCs, such as mid to posterior HOXA genes, may be activated with partial CTR9 loss. Remarkably, we observed a significant enrichment for hematopoietic SEC target genes upon CTR9 perturbation in HSCs by gene set enrichment analysis (normalized enrichment score = 3.29, p-value < 0.001). In light of these findings suggesting that SEC activity may be increased with partial CTR9 loss-of-function, as occurs in individuals harboring myeloid malignancy variants, we sought to functionally validate these observations. Using the inhibitors of the SEC, including SR-0813 that targets MLLT3 or with an inhibitor of CDK9, we noted rescue of the CTR9-mediated expansion of phenotypic LT- and ST-HSCs without a significant impact on the bulk HSPC population. To further elucidate underlying mechanisms, we performed immunoprecipitation of PAF1 or SEC component MLLT3 in HSPCs with control or CTR9 editing. While we continued to pull down all PAF1 complex components with PAF1, we also noted pulldown of MLLT3, which increased with CTR9 editing. MLLT3 immunoprecipitation revealed selective pulldowns of PAF1 and CDC73, which also increased with CTR9 editing. These findings show how PAF1 complex components PAF1 and CDC73 interact with and stimulate SEC activity. Our findings reveal how CTR9 usually restricts this activity and constrains transcriptional elongation to limit HSC self-renewal. We functionally validated these findings through selective editing of different PAF1 complex components in HSPCs: we observed reduced HSCs upon editing of PAF1 and CDC73, but increases with editing of other PAF1 complex components. Our findings collectively demonstrate a mechanism by which a previously undefined myeloid malignancy predisposition occurs. We demonstrate that CTR9 loss-of-function stimulates SEC activity and thereby results in HSC expansion to confer risk for acquiring MPNs and other myeloid malignancies. Disclosures Armstrong: Neomorph Inc: Consultancy, Current holder of individual stocks in a privately-held company; Imago Biosciences: Consultancy; Vitae/Allergan Pharma: Consultancy; Cyteir Therapeutics: Consultancy; C4 Therapeutics: Consultancy; OxStem Oncology: Consultancy; Accent Therapeutics: Consultancy; Mana Therapeutics: Consultancy; Janssen: Research Funding; Novartis: Research Funding; Syndax: Research Funding; AstraZeneca: Research Funding. Sankaran: Ensoma: Consultancy; Forma: Consultancy; Cellarity: Consultancy; Novartis: Consultancy; Branch Biosciences: Consultancy.

Follow-up Next Generation Sequencing (NGS) Analyses on Clonal Chronic Idiopathic Neutropenia (CIN) Patients: Insights in the Natural History of the Disease

Background: We have previously performed NGS analysis of genes that are recurrently mutated in myeloid malignancies in a cohort of patients with the diagnosis of chronic idiopathic neutropenia (CIN) according to previously reported criteria that largely overlap with those proposed for idiopathic cytopenia/neutropenia of undetermined significance (ICUS-N). We have thus estimated for the first time the frequency of clonal hematopoiesis in patients with CIN/ICUS-N (11.54%) and found that clonal CIN patients have a significantly higher risk of developing a myeloid neoplasm than those with no evidence of clonality (non-clonal). 1 However more longitudinal follow-up NGS studies are required for the tracking of clonal evolution and delineation of clonal CIN natural history. Aims: To conduct longitudinal follow-up NGS analyses in order to assess clonal evolution and associate the clinical significance of detected clonal aberrations with the risk of transforming to myeloid malignancy in clonal CIN clinical outcome. Methods: Genomic DNA was extracted from patients' BM or PB samples, sequencing libraries were prepared and subjected to targeted next generation sequencing (NGS) on an Ion S5 Prime Sequencer (Thermo Fisher Scientific) using a panel of 38 genes recurrently mutated in myeloid malignancies. Results: Follow-up analysis by NGS was performed in 16 clonal CIN patients (Figure 1). (Out of these 16 patients, follow-up NGS data has already been published in 9 patients, however additional timepoints were tested in 3 of them). 1 The median time between the first and subsequent analysis was 28.5 months (range 8-164 months). Ten of these patients carried the initial somatic mutations with only subtle changes in the size of clone as estimated by the variant allele frequency (VAF); the patients displayed absence of additional mutations and did not develop myeloid malignancy (Figure 1A-C, E-G, I, K, L, P). Two patients acquired a second mutation at follow-up. One of them still displayed stable disease course (Figure 1D) whereas the second eventually progressed to CMML (Figure 1H). The analysis also revealed that one patient lost the initial detected mutation at follow-up after 98 months (Figure 1J). Two patients who progressed to MDS/MPN and AML respectively, displayed a notable clonal expansion with additional mutations at the time of progression (Figure 1M and Figure 1N, respectively). Specifically, the patient who progressed to MDS/MPN acquired a mutation in JAK2 and ASXL1 while the patient who progressed to AML acquired the typical NPM1 p.L287fs mutation. The patient who developed MDS with multilineage dysplasia, carrying three mutations in DNMT3A and IDH1, showed a moderate increase in the VAF of these mutations at first follow-up (Figure 1O). The patient progressed to acute lymphoblastic leukemia (2 nd follow-up) with acquisition of additional truncating mutation in ETV6. Following treatment (3 rd and 4 th follow-up) mutation in ETV6 was lost, however the three mutations in DNMT3A and IDH1 persisted and their clone size increased. Conclusions: In the majority of patients tested for clonal evolution over time, most mutant clones appeared to be remarkably stable, with minimal VAF change, no acquisition of new molecular alterations and no progression to overt myeloid malignancy. Two CIN patients who transformed to a myeloid malignancy displayed a clonal expansion as was reflected by the increase of VAF and the development of additional mutations whereas in the third patient only a modest VAF increase was identified before malignant transformation. Finally in the patient bearing 4 mutations no progression to overt malignancy was observed after 12 months of follow-up. This ongoing study of sequential NGS analysis of CIN patients is anticipated to contribute to the better understanding and enrich further the knowledge on the natural history of this rare disease. References: Tsaknakis G, Galli A, Papadakis S et al. Incidence and Prognosis of Clonal Hematopoiesis in patients with Chronic Idiopathic Neutropenia. Blood. 2021 Jun 24:blood.2021010815. doi: 10.1182/blood.2021010815. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.