Enhanced Molecular Response in Myeloproliferative Neoplasms with Complete JAK2V617F Inhibition.

SUMMARY: Dunbar, Bowman, and colleagues present here a novel genetic mouse model with inducible and reversible expression of the JAK2V617F mutation in the endogenous locus. Results from this study clearly demonstrate an absolute requirement for myeloproliferative neoplasm-initiating cells for this mutation in their survival and imply that more efficacious inhibitors could be curative for these patients even in the setting of additional cooperating mutations. See related article by Dunbar et al., p. 737 (8).

Spatial Mapping of Hematopoietic Clones in Human Bone Marrow.

Clonal hematopoiesis (CH) is the expansion of somatically mutated cells in the hematopoietic compartment of individuals without hematopoietic dysfunction. Large CH clones (i.e., >2% variant allele fraction) predispose to hematologic malignancy, but CH is detected at lower levels in nearly all middle-aged individuals. Prior work has extensively characterized CH in peripheral blood, but the spatial distribution of hematopoietic clones in human bone marrow is largely undescribed. To understand CH at this level, we developed a method for spatially aware somatic mutation profiling and characterized the bone marrow of a patient with polycythemia vera. We identified the complex clonal distribution of somatic mutations in the hematopoietic compartment, the restriction of somatic mutations to specific subpopulations of hematopoietic cells, and spatial constraints of these clones in the bone marrow. This proof of principle paves the way to answering fundamental questions regarding CH spatial organization and factors driving CH expansion and malignant transformation in the bone marrow. SIGNIFICANCE: CH occurs commonly in humans and can predispose to hematologic malignancy. Although well characterized in blood, it is poorly understood how clones are spatially distributed in the bone marrow. To answer this, we developed methods for spatially aware somatic mutation profiling to describe clonal heterogeneity in human bone marrow. See related commentary by Austin and Aifantis, p. 139.

Droplet Digital PCR for Oncogenic KMT2A Fusion Detection.

Acute myeloid leukemia (AML) is an aggressive blood cancer diagnosed in approximately 120,000 individuals worldwide each year. During treatment for AML, detecting residual disease is essential for prognostication and treatment decision-making. Currently, methods for detecting residual AML are limited to identifying approximately 1:100 to 1:1000 leukemic cells (morphology and DNA sequencing) or are difficult to implement (flow cytometry). AML arising after chemotherapy or radiation exposure is termed therapy-related AML (t-AML) and is exceptionally aggressive and treatment resistant. t-AML is often driven by oncogenic fusions that result from prior treatments that introduce double-strand DNA breaks. The most common t-AML-associated translocations affect KMT2A. There are at least 80 known KMT2A fusion partners, but approximately 80% of fusions involve only five partners-AF9, AF6, AF4, ELL, and ENL. We present a novel droplet digital PCR assay targeting the most common KMT2A-rearrangements to enable detection of rare AML cells harboring these fusions. This assay was benchmarked in cell lines and patient samples harboring oncogenic KMT2A fusions and demonstrated a limit of detection of approximately 1:1,000,000 cells. Future application of this assay could improve disease detection and treatment decision-making for patients with t-AML with KMT2A fusions and premalignant oncogenic fusion detection in at-risk individuals after chemotherapy exposure.

KDM6B protects T-ALL cells from NOTCH1-induced oncogenic stress.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic neoplasm resulting from the malignant transformation of T-cell progenitors. While activating NOTCH1 mutations are the dominant genetic drivers of T-ALL, epigenetic dysfunction plays a central role in the pathology of T-ALL and can provide alternative mechanisms to oncogenesis in lieu of or in combination with genetic mutations. The histone demethylase enzyme KDM6A (UTX) is also recurrently mutated in T-ALL patients and functions as a tumor suppressor. However, its gene paralog, KDM6B (JMJD3), is never mutated and can be significantly overexpressed, suggesting it may be necessary for sustaining the disease. Here, we used mouse and human T-ALL models to show that KDM6B is required for T-ALL development and maintenance. Using NOTCH1 gain-of-function retroviral models, mouse cells genetically deficient for Kdm6b were unable to propagate T-ALL. Inactivating KDM6B in human T-ALL patient cells by CRISPR/Cas9 showed KDM6B-targeted cells were significantly outcompeted over time. The dependence of T-ALL cells on KDM6B was proportional to the oncogenic strength of NOTCH1 mutation, with KDM6B required to prevent stress-induced apoptosis from strong NOTCH1 signaling. These studies identify a crucial role for KDM6B in sustaining NOTCH1-driven T-ALL and implicate KDM6B as a novel therapeutic target in these patients.

DUSP6 mediates resistance to JAK2 inhibition and drives leukemic progression.

Myeloproliferative neoplasms (MPNs) exhibit a propensity for transformation to secondary acute myeloid leukemia (sAML), for which the underlying mechanisms remain poorly understood, resulting in limited treatment options and dismal clinical outcomes. Here, we performed single-cell RNA sequencing on serial MPN and sAML patient stem and progenitor cells, identifying aberrantly increased expression of DUSP6 underlying disease transformation. Pharmacologic dual-specificity phosphatase (DUSP)6 targeting led to inhibition of S6 and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling while also reducing inflammatory cytokine production. DUSP6 perturbation further inhibited ribosomal S6 kinase (RSK)1, which we identified as a second indispensable candidate associated with poor clinical outcome. Ectopic expression of DUSP6 mediated JAK2-inhibitor resistance and exacerbated disease severity in patient-derived xenograft (PDX) models. Contrastingly, DUSP6 inhibition potently suppressed disease development across Jak2V617F and MPLW515L MPN mouse models and sAML PDXs without inducing toxicity in healthy controls. These findings underscore DUSP6 in driving disease transformation and highlight the DUSP6-RSK1 axis as a vulnerable, druggable pathway in myeloid malignancies.

Distinct Tumor Necrosis Factor Alpha Receptors Dictate Stem Cell Fitness versus Lineage Output in Dnmt3a-Mutant Clonal Hematopoiesis.

Clonal hematopoiesis resulting from the enhanced fitness of mutant hematopoietic stem cells (HSC) associates with both favorable and unfavorable health outcomes related to the types of mature mutant blood cells produced, but how this lineage output is regulated is unclear. Using a mouse model of a clonal hematopoiesis-associated mutation, DNMT3AR882/+ (Dnmt3aR878H/+), we found that aging-induced TNFα signaling promoted the selective advantage of mutant HSCs and stimulated the production of mutant B lymphoid cells. The genetic loss of the TNFα receptor TNFR1 ablated the selective advantage of mutant HSCs without altering their lineage output, whereas the loss of TNFR2 resulted in the overproduction of mutant myeloid cells without altering HSC fitness. These results nominate TNFR1 as a target to reduce clonal hematopoiesis and the risk of associated diseases and support a model in which clone size and mature blood lineage production can be independently controlled to modulate favorable and unfavorable clonal hematopoiesis outcomes. SIGNIFICANCE: Through the identification and dissection of TNFα signaling as a key driver of murine Dnmt3a-mutant hematopoiesis, we report the discovery that clone size and production of specific mature blood cell types can be independently regulated. See related commentary by Niño and Pietras, p. 2724. This article is highlighted in the In This Issue feature, p. 2711.

Effect of Clonal Hematopoiesis on Cardiovascular Disease in People Living with HIV.

Hematopoietic stem cells (HSCs) with age-associated somatic mutations that disproportionally contribute to hematopoiesis generate the condition known as clonal hematopoiesis (CH). While CH conveys increased risk of hematologic cancer, there is also strong association between CH and cardiovascular disease (CVD). Accumulating evidence suggests that inflammation mechanistically links CH to CVD, and we hypothesized that CH may be a predictive biomarker of CVD in conditions of chronic inflammation. One such patient population comprises people living with HIV (PLWH) who also have substantially increased incidences of CVD and CH . We studied the association between CH and CVD in PLWH using samples from ACTG Study A5001 (or ALLRT), a prospective clinical trial of HIV-infected persons with long-term follow-up. We observed a positive association between CH and CVD in PLWH independent of traditional CVD risk factors. Moreover, in CVD cases, the CH clone was identifiable in the blood years before CVD diagnosis, unlike in PLWH with CH who did not have CVD. With the life span of PLWH increasing because of advances in treatment, our results indicate that the presence of CH and its clonal dynamics could be used as a prognostic biomarker of the risk for CVD in PLWH.

Txnip Enhances Fitness of Dnmt3a-Mutant Hematopoietic Stem Cells via p21.

Clonal hematopoiesis (CH) refers to the age-related expansion of specific clones in the blood system, and manifests from somatic mutations acquired in hematopoietic stem cells (HSCs). Most CH variants occur in the gene DNMT3A, but while DNMT3A-mutant CH becomes almost ubiquitous in aging humans, a unifying molecular mechanism to illuminate how DNMT3A-mutant HSCs outcompete their counterparts is lacking. Here, we used interferon gamma (IFNγ) as a model to study the mechanisms by which Dnmt3a mutations increase HSC fitness under hematopoietic stress. We found Dnmt3a-mutant HSCs resist IFNγ-mediated depletion, and IFNγ-signaling is required for clonal expansion of Dnmt3a-mutant HSCs in vivo. Mechanistically, DNA hypomethylation-associated overexpression of Txnip in Dnmt3a-mutant HSCs leads to p53 stabilization and upregulation of p21. This preserves the functional potential of Dnmt3a-mutant HSCs through increased quiescence and resistance to IFNγ-induced apoptosis. These data identify a previously undescribed mechanism to explain increased fitness of DNMT3A-mutant clones under hematopoietic stress. SIGNIFICANCE: DNMT3A mutations are common variants in clonal hematopoiesis, and recurrent events in blood cancers. Yet the mechanisms by which these mutations provide hematopoietic stem cells a competitive advantage as a precursor to malignant transformation remain unclear. Here, we use inflammatory stress to uncover molecular mechanisms leading to this fitness advantage.See related commentary by De Dominici and DeGregori, p. 178. This article is highlighted in the In This Issue feature, p. 171.

Pevonedistat targets malignant cells in myeloproliferative neoplasms in vitro and in vivo via NFκB pathway inhibition.

Targeted inhibitors of JAK2 (eg ruxolitinib) often provide symptomatic relief for myeloproliferative neoplasm (MPN) patients, but the malignant clone persists and remains susceptible to disease transformation. These observations suggest that targeting alternative dysregulated signaling pathways may provide therapeutic benefit. Previous studies identified NFκB pathway hyperactivation in myelofibrosis (MF) and secondary acute myeloid leukemia (sAML) that was insensitive to JAK2 inhibition. Here, we provide evidence that NFκB pathway inhibition via pevonedistat targets malignant cells in MPN patient samples as well as in MPN and patient-derived xenograft mouse models that are nonredundant with ruxolitinib. Colony forming assays revealed preferential inhibition of MF colony growth compared with normal colony formation. In mass cytometry studies, pevonedistat blunted canonical TNFα responses in MF and sAML patient CD34+ cells. Pevonedistat also inhibited hyperproduction of inflammatory cytokines more effectively than ruxolitinib. Upon pevonedistat treatment alone or in combination with ruxolitinib, MPN mouse models exhibited reduced disease burden and improved survival. These studies demonstrating efficacy of pevonedistat in MPN cells in vitro as well as in vivo provide a rationale for therapeutic inhibition of NFκB signaling for MF treatment. Based on these findings, a Phase 1 clinical trial combining pevonedistat with ruxolitinib has been initiated.

Simplified murine multipotent progenitor isolation scheme: Establishing a consensus approach for multipotent progenitor identification.

The mouse hematopoietic system has served as a paradigm for analysis of developmental fate decisions in tissue homeostasis and regeneration. However, multiple immunophenotypic definitions of, and sometimes divergent nomenclatures used to classify, murine multipotent progenitors (MPPs) have emerged in the field over time. This has created significant confusion and inconsistency in the hematology field. To facilitate easier comparison of murine MPP phenotypes between research laboratories, a working group of four International Society for Experimental Hematology (ISEH) members with extensive experience studying the functional activities associated with different MPP phenotypic definitions reviewed the current state of the field with the goal of developing a position statement toward a simplified and unified immunophenotypic definition of MPP populations. In November of 2020, this position statement was presented as a webinar to the ISEH community for discussion and feedback. Hence, the Simplified MPP Identification Scheme presented here is the result of curation of existing literature, consultation with leaders in the field, and crowdsourcing from the wider experimental hematology community. Adoption of a unified definition and nomenclature, while still leaving room for individual investigator customization, will benefit scientists at all levels trying to compare these populations between experimental settings.

A Humanized Animal Model Predicts Clonal Evolution and Therapeutic Vulnerabilities in Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPN) are chronic blood diseases with significant morbidity and mortality. Although sequencing studies have elucidated the genetic mutations that drive these diseases, MPNs remain largely incurable with a significant proportion of patients progressing to rapidly fatal secondary acute myeloid leukemia (sAML). Therapeutic discovery has been hampered by the inability of genetically engineered mouse models to generate key human pathologies such as bone marrow fibrosis. To circumvent these limitations, here we present a humanized animal model of myelofibrosis (MF) patient-derived xenografts (PDX). These PDXs robustly engrafted patient cells that recapitulated the patient's genetic hierarchy and pathologies such as reticulin fibrosis and propagation of MPN-initiating stem cells. The model can select for engraftment of rare leukemic subclones to identify patients with MF at risk for sAML transformation and can be used as a platform for genetic target validation and therapeutic discovery. We present a novel but generalizable model to study human MPN biology. SIGNIFICANCE: Although the genetic events driving MPNs are well defined, therapeutic discovery has been hampered by the inability of murine models to replicate key patient pathologies. Here, we present a PDX system to model human myelofibrosis that reproduces human pathologies and is amenable to genetic and pharmacologic manipulation. This article is highlighted in the In This Issue feature, p. 2945.

TET2 and DNMT3A Mutations Exert Divergent Effects on DNA Repair and Sensitivity of Leukemia Cells to PARP Inhibitors.

Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that affect the epigenetic regulation of DNA methylation. Mutations in both genes often co-occur with activating mutations in genes encoding oncogenic tyrosine kinases such as FLT3ITD, BCR-ABL1, JAK2V617F , and MPLW515L , or with mutations affecting related signaling pathways such as NRASG12D and CALRdel52 . Here, we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing these oncogenes. Malignant TET2-deficient cells displayed downregulation of BRCA1 and LIG4, resulting in reduced activity of BRCA1/2-mediated homologous recombination (HR) and DNA-PK-mediated non-homologous end-joining (D-NHEJ), respectively. TET2-deficient cells relied on PARP1-mediated alternative NHEJ (Alt-NHEJ) for protection from the toxic effects of spontaneous and drug-induced DNA double-strand breaks. Conversely, DNMT3A-deficient cells favored HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells were sensitive to PARP inhibitor (PARPi) treatment in vitro and in vivo, whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity or TET2-Wilms' tumor 1 (WT1)-binding ability was responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Collectively, these findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. SIGNIFICANCE: TET2 and DNMT3A mutations affect distinct DNA repair mechanisms and govern the differential sensitivities of oncogenic tyrosine kinase-positive malignant hematopoietic cells to PARP inhibitors.

Epigenomic regulation of human T-cell leukemia virus by chromatin-insulator CTCF.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes an aggressive T-cell malignancy and a variety of inflammatory conditions. The integrated provirus includes a single binding site for the epigenomic insulator, CCCTC-binding protein (CTCF), but its function remains unclear. In the current study, a mutant virus was examined that eliminates the CTCF-binding site. The mutation did not disrupt the kinetics and levels of virus gene expression, or establishment of or reactivation from latency. However, the mutation disrupted the epigenetic barrier function, resulting in enhanced DNA CpG methylation downstream of the CTCF binding site on both strands of the integrated provirus and H3K4Me3, H3K36Me3, and H3K27Me3 chromatin modifications both up- and downstream of the site. A majority of clonal cell lines infected with wild type HTLV-1 exhibited increased plus strand gene expression with CTCF knockdown, while expression in mutant HTLV-1 clonal lines was unaffected. These findings indicate that CTCF binding regulates HTLV-1 gene expression, DNA and histone methylation in an integration site dependent fashion.

Chronic infection drives Dnmt3a-loss-of-function clonal hematopoiesis via IFNγ signaling.

Age-related clonal hematopoiesis (CH) is a risk factor for malignancy, cardiovascular disease, and all-cause mortality. Somatic mutations in DNMT3A are drivers of CH, but decades may elapse between the acquisition of a mutation and CH, suggesting that environmental factors contribute to clonal expansion. We tested whether infection provides selective pressure favoring the expansion of Dnmt3a mutant hematopoietic stem cells (HSCs) in mouse chimeras. We created Dnmt3a-mosaic mice by transplanting Dnmt3a-/- and WT HSCs into WT mice and observed the substantial expansion of Dnmt3a-/- HSCs during chronic mycobacterial infection. Injection of recombinant IFNγ alone was sufficient to phenocopy CH by Dnmt3a-/- HSCs upon infection. Transcriptional and epigenetic profiling and functional studies indicate reduced differentiation associated with widespread methylation alterations, and reduced secondary stress-induced apoptosis accounts for Dnmt3a-/- clonal expansion during infection. DNMT3A mutant human HSCs similarly exhibit defective IFNγ-induced differentiation. We thus demonstrate that IFNγ signaling induced during chronic infection can drive DNMT3A-loss-of-function CH.

LKB1/STK11 Is a Tumor Suppressor in the Progression of Myeloproliferative Neoplasms.

The myeloproliferative neoplasms (MPN) frequently progress to blast phase disease, an aggressive form of acute myeloid leukemia. To identify genes that suppress disease progression, we performed a focused CRISPR/Cas9 screen and discovered that depletion of LKB1/Stk11 led to enhanced in vitro self-renewal of murine MPN cells. Deletion of Stk11 in a mouse MPN model caused rapid lethality with enhanced fibrosis, osteosclerosis, and an accumulation of immature cells in the bone marrow, as well as enhanced engraftment of primary human MPN cells in vivo. LKB1 loss was associated with increased mitochondrial reactive oxygen species and stabilization of HIF1α, and downregulation of LKB1 and increased levels of HIF1α were observed in human blast phase MPN specimens. Of note, we observed strong concordance of pathways that were enriched in murine MPN cells with LKB1 loss with those enriched in blast phase MPN patient specimens, supporting the conclusion that STK11 is a tumor suppressor in the MPNs. SIGNIFICANCE: Progression of the myeloproliferative neoplasms to acute myeloid leukemia occurs in a substantial number of cases, but the genetic basis has been unclear. We discovered that loss of LKB1/STK11 leads to stabilization of HIF1a and promotes disease progression. This observation provides a potential therapeutic avenue for targeting progression.This article is highlighted in the In This Issue feature, p. 1307.

TGFßR-SMAD3 Signaling Induces Resistance to PARP Inhibitors in the Bone Marrow Microenvironment.

Synthetic lethality triggered by PARP inhibitor (PARPi) yields promising therapeutic results. Unfortunately, tumor cells acquire PARPi resistance, which is usually associated with the restoration of homologous recombination, loss of PARP1 expression, and/or loss of DNA double-strand break (DSB) end resection regulation. Here, we identify a constitutive mechanism of resistance to PARPi. We report that the bone marrow microenvironment (BMM) facilitates DSB repair activity in leukemia cells to protect them against PARPi-mediated synthetic lethality. This effect depends on the hypoxia-induced overexpression of transforming growth factor beta receptor (TGFßR) kinase on malignant cells, which is activated by bone marrow stromal cells-derived transforming growth factor beta 1 (TGF-ß1). Genetic and/or pharmacological targeting of the TGF-ß1-TGFßR kinase axis results in the restoration of the sensitivity of malignant cells to PARPi in BMM and prolongs the survival of leukemia-bearing mice. Our finding may lead to the therapeutic application of the TGFßR inhibitor in patients receiving PARPis.

Clonal hematopoiesis: mechanisms driving dominance of stem cell clones.

The discovery of clonal hematopoiesis (CH) in older individuals has changed the way hematologists and stem cell biologists view aging. Somatic mutations accumulate in stem cells over time. While most mutations have no impact, some result in subtle functional differences that ultimately manifest in distinct stem cell behaviors. With a large pool of stem cells and many decades to compete, some of these differences confer advantages under specific contexts. Approximately 20 genes are recurrently found as mutated in CH, indicating they confer some advantage. The impact of these mutations has begun to be analyzed at a molecular level by modeling in cell lines and in mice. Mutations in epigenetic regulators such as DNMT3A and TET2 confer an advantage by enhancing self-renewal of stem and progenitor cells and inhibiting their differentiation. Mutations in other genes involved in the DNA damage response may simply enhance cell survival. Here, we review proposed mechanisms that lead to CH, specifically in the context of stem cell biology, based on our current understanding of the function of some of the CH-associated genes.

Radiation induces iatrogenic immunosuppression by indirectly affecting hematopoiesis in bone marrow.

The immune system plays a vital role in cancer therapy, especially with the advent of immunotherapy. Radiation therapy induces iatrogenic immunosuppression referred to as radiation-induced lymphopenia (RIL). RIL correlates with significant decreases in the overall survival of cancer patients. Although the etiology and severity of lymphopenia are known, the mechanism(s) of RIL are largely unknown. We found that irradiation not only had direct effects on circulating lymphocytes but also had indirect effects on the spleen, thymus, and bone marrow. We found that irradiated cells traffic to the bone marrow and bring about the reduction of hematopoietic stem cells (HSC) and progenitor cells. Using mass cytometry analysis (CyTOF) of the bone marrow, we found reduced expression of CD11a, which is required for T cell proliferation and maturation. RNA Sequencing and gene set enrichment analysis of the bone marrow cells following irradiation showed down-regulation of genes involved in hematopoiesis. Identification of CD11a and hematopoietic genes involved in iatrogenic immune suppression can help identify mechanisms of RIL.

Divergent Effects of Dnmt3a and Tet2 Mutations on Hematopoietic Progenitor Cell Fitness.

The DNA methylation regulators DNMT3A and TET2 are recurrently mutated in hematological disorders. Despite possessing antagonistic biochemical activities, loss-of-function murine models show overlapping phenotypes in terms of increased hematopoietic stem cell (HSC) fitness. Here, we directly compared the effects of these mutations on hematopoietic progenitor function and disease initiation. In contrast to Dnmt3a-null HSCs, which possess limitless self-renewal in vivo, Tet2-null HSCs unexpectedly exhaust at the same rate as control HSCs in serial transplantation assays despite an initial increase in self-renewal. Moreover, loss of Tet2 more acutely sensitizes hematopoietic cells to the addition of a common co-operating mutation (Flt3ITD) than loss of Dnmt3a, which is associated with a more rapid expansion of committed progenitor cells. The effect of Tet2 mutation manifests more profound myeloid lineage skewing in committed hematopoietic progenitor cells rather than long-term HSCs. Molecular characterization revealed divergent transcriptomes and chromatin accessibility underlying these functional differences.