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•DNA POLθ–mediated DNA repair protects leukemia cells from the toxic effect of metabolically induced DNA damage.•DNA POLθ is a new therapeutic target to eradicate leukemia clones expressing oncogenic tyrosine kinases.

Superoxide dismutase 2 (SOD2) catalyzes the dismutation of superoxide to hydrogen peroxide in mitochondria, limiting mitochondrial damage. The SOD2 amino acid valine-to-alanine substitution at position 16 (V16A) in the mitochondrial leader sequence is a common genetic variant among patients with sickle cell disease (SCD). However, little is known about the cardiovascular consequences of SOD2V16A in SCD patients or its impact on endothelial cell function. Here, we show SOD2V16A associates with increased tricuspid regurgitant velocity (TRV), systolic blood pressure, right ventricle area at systole, and declined 6-minute walk distance in 410 SCD patients. Plasma lactate dehydrogenase, a marker of oxidative stress and hemolysis, significantly associated with higher TRV. To define the impact of SOD2V16A in the endothelium, we introduced the SOD2V16A variant into endothelial cells. SOD2V16A increases hydrogen peroxide and mitochondrial reactive oxygen species (ROS) production compared with controls. Unexpectedly, the increased ROS was not due to SOD2V16A mislocalization but was associated with mitochondrial complex IV and a concomitant decrease in basal respiration and complex IV activity. In sum, SOD2V16A is a novel clinical biomarker of cardiovascular dysfunction in SCD patients through its ability to decrease mitochondrial complex IV activity and amplify ROS production in the endothelium.

Somatic gene mutations are key determinants of outcome in patients with myelodysplastic syndromes (MDS) and secondary AML (sAML). In particular, patients with TP53 mutations represent a distinct molecular cohort with uniformly poor prognosis. The precise pathogenetic mechanisms underlying these inferior outcomes have not been delineated. In this study, we characterized the immunological features of the malignant clone and alterations in the immune microenvironment in patients with TP53-mutant and wild-type MDS or sAML. Notably, PDL1 expression is significantly increased in hematopoietic stem cells of patients with TP53 mutations, which is associated with MYC upregulation and marked downregulation of MYC’s negative regulator miR-34a, a p53 transcription target. Notably, patients with TP53 mutations display significantly reduced numbers of bone marrow–infiltrating OX40+ cytotoxic T cells and helper T cells, as well as decreased ICOS+ and 4-1BB+ natural killer cells. Further, highly immunosuppressive regulatory T cells (Tregs) (ie, ICOShigh/PD-1−) and myeloid-derived suppressor cells (PD-1low) are expanded in cases with TP53 mutations. Finally, a higher proportion of bone marrow–infiltrating ICOShigh/PD-1− Treg cells is a highly significant independent predictor of overall survival. We conclude that the microenvironment of TP53 mutant MDS and sAML has an immune-privileged, evasive phenotype that may be a primary driver of poor outcomes and submit that immunomodulatory therapeutic strategies may offer a benefit for this molecularly defined subpopulation.

Somatic gene mutations are key determinants of outcome in patients with myelodysplastic syndromes (MDS) and secondary AML (sAML). In particular, patients with TP53mutations represent a distinct molecular cohort with uniformly poor prognosis. The precise pathogenetic mechanisms underlying these inferior outcomes have not been delineated. In this study, we characterized the immunological features of the malignant clone and alterations in the immune microenvironment in patients with TP53-mutant and wild-type MDS or sAML. Notably, PDL1 expression is significantly increased in hematopoietic stem cells of patients with TP53mutations, which is associated with MYCupregulation and marked downregulation of MYC's negative regulator miR-34a, a p53 transcription target. Notably, patients with TP53mutations display significantly reduced numbers of bone marrow–infiltrating OX40+cytotoxic T cells and helper T cells, as well as decreased ICOS+and 4-1BB+natural killer cells. Further, highly immunosuppressive regulatory T cells (Tregs) (ie, ICOShigh/PD-1−) and myeloid-derived suppressor cells (PD-1low) are expanded in cases with TP53mutations. Finally, a higher proportion of bone marrow–infiltrating ICOShigh/PD-1−Treg cells is a highly significant independent predictor of overall survival. We conclude that the microenvironment of TP53mutant MDS and sAML has an immune-privileged, evasive phenotype that may be a primary driver of poor outcomes and submit that immunomodulatory therapeutic strategies may offer a benefit for this molecularly defined subpopulation.

Wiskott-Aldrich syndrome (WAS) is an X-linked disease caused by mutations in the WAS gene, leading to thrombocytopenia, eczema, recurrent infections, autoimmune disease, and malignancy. Hematopoietic cell transplantation (HCT) is the primary curative approach, with the goal of correcting the underlying immunodeficiency and thrombocytopenia. HCT outcomes have improved over time, particularly for patients with HLA-matched sibling and unrelated donors. We report the outcomes of 129 patients with WAS who underwent HCT at 29 Primary Immune Deficiency Treatment Consortium centers from 2005 through 2015. Median age at HCT was 1.2 years. Most patients (65%) received myeloablative busulfan-based conditioning. With a median follow-up of 4.5 years, the 5-year overall survival (OS) was 91%. Superior 5-year OS was observed in patients <5 vs ≥5 years of age at the time of HCT (94% vs 66%; overall P = .0008). OS was excellent regardless of donor type, even in cord blood recipients (90%). Conditioning intensity did not affect OS, but was associated with donor T-cell and myeloid engraftment after HCT. Specifically, patients who received fludarabine/melphalan-based reduced-intensity regimens were more likely to have donor myeloid chimerism <50% early after HCT. In addition, higher platelet counts were observed among recipients who achieved full (>95%) vs low-level (5%-49%) donor myeloid engraftment. In summary, HCT outcomes for WAS have improved since 2005, compared with prior reports. HCT at a younger age continues to be associated with superior outcomes supporting the recommendation for early HCT. High-level donor myeloid engraftment is important for platelet reconstitution after either myeloablative or busulfan-containing reduced intensity conditioning. (This trial was registered at www.clinicaltrials.gov as #NCT02064933.)

Approximately 35% to 50% of patients otherwise cured of hematologic malignancies after allogeneic hematopoietic stem cell transplantation will develop the pleomorphic autoimmune-like syndrome known as chronic graft-versus-host disease (cGVHD). Since in 2005, National Institutes of Health (NIH) consensus panels have proposed definitions and classifications of disease to standardize treatment trials. Recently, the first agent was approved by the US Food and Drug Administration for steroid-refractory cGVHD. Despite these advances, most individuals do not achieve durable resolution of disease activity with initial treatment. Moreover, standardized recommendations on how to best implement existing and novel immunomodulatory agents and taper salvage agents are often lacking. Given the potential life-threatening nature of cGVHD, we employ in our practice patient assessment templates at each clinic visit to elucidate known prognostic indicators and red flags. We find NIH scoring templates practical for ongoing assessments of these complex patient cases and determination of when changes in immunosuppressive therapy are warranted. Patients not eligible or suitable for clinical trials have systemic and organ-directed adjunctive treatments crafted in a multidisciplinary clinic. Herein, we review these treatment options and offer a management and monitoring scaffold for representative patients with cGVHD not responding to initial therapy.

Background

Progression of myeloproliferative neoplasms to blast phase (BPMPN) is associated with lack of response to conventional therapies and dire clinical outcomes. Consequently, there is a major unmet need to develop new therapies for BPMPN. Chromothripsis, the process of catastrophic shattering and haphazard repair of chromosomes, is a key contributor to somatic variation in cancer, but this phenomenon has not yet been described in BPMPN. More broadly, whether chromothripsis might result in actionable molecular events that are amenable to targeting remains an open question.

Introduction:The burden of the management and clinical sequelae of hemophilia B (HB) negatively impacts health-related quality of life (HRQoL), including chronic pain and mental health. Fidanacogene elaparvovec (PF-06838435, formerly SPK-9001) is an adeno-associated virus-based gene therapy vector transferring the high activity variant of human factor IX (FIX), FIX-R338L, aimed at enabling endogenous FIX expression in individuals with hemophilia B. We present descriptive data on the impact on HRQoL for participants in the fidanacogene elaparvovec phase 1/2a study.

INTRODUCTION

SF3B1is the most prevalent splicing factor mutated in myeloid neoplasms, detected in 20-30% of patients with myelodysplasia (MDS) and associated with a milder disease phenotype. SF3B1mutations are typically single nucleotide variations occurring in hotspots in the HEAT domain. Disease-specific SF3B1hotspot predilection is observed; for example, K700E is most common in MDS and R625 in uveal melanoma. The K666 hotspot has been associated with increased risk of MDS disease progression to acute myeloid leukemia (AML). However, the prognostic impact across all myeloid neoplasms inclusive of myeloproliferative neoplasms (MPN) and MDS/MPN overlap syndromes has not been established.

Cytogenetic methods are a cornerstone of the modern diagnostic workflow for acute myeloid leukemia and related disorders. The goal of these methods is to identify structural genomic aberrations that drive the cancer or otherwise act as an indicator of patient risk. Despite their success, any one cytogenetic test is limited either in resolution, ability to provide unbiased survey of the genome, or ability to detect balanced aberrations. To address these limitations, we have applied proximity ligation sequencing (PLS) to characterize structural variants (SV) in AML genomes. PLS captures ultra-long-range sequence information without high-molecular-weight DNA and requires a modest (5-7x) sequence coverage of the genome. Applying this method to AML diagnostic specimens, we find that PLS identifies cytogenetically defined translocations (reciprocal and non-reciprocal) and inversions with specificity and sensitivity >0.95. The high resolution of PLS (<10kb) detected non-canonical variants missed by standard methods as well as structural variants below limits of detection by karyotyping, identifying variants in over half the patients previously determined to have a normal karyotype (Table 1). This included previously described variants of clinical significance as well as a recurrent inversion not previously described in AML. These observations highlight the effectiveness of PLS to provide high-resolution insights into known clinically relevant variants and discover variants missed by traditional methodologies.

Myeloid malignant cells (AML, MPN, CML) expressing oncogenic tyrosine kinases [OTKs: FLT3(ITD), JAK2(V617F), BCR/ABL1, respectively] accumulate DNA damage but altered DNA repair mechanisms protect them from apoptosis. We reported before that formaldehyde generated by altered serine/one-carbon cycle metabolism in malignant cells harboring OTKs contributed to accumulation of toxic DNA-protein crosslinks (DPCs) which were converted to highly lethal DNA double-strand breaks (DSBs). To counteract the toxicity of DSBs, OTKs enhanced the expression of DNA polymerase theta (PolΘ, encoded by POLQgene), a unique DNA helicase-DNA polymerase fusion protein that promotes error-prone repair of DSBs by a mechanism referred to as PolΘ-mediated DNA end-joining (TMEJ). PolΘ plays an essential role in the initiation and maintenance of hematological malignancies harboring OTKs. Although the cellular activities of PolΘ have been widely studied, little is known about how PolΘ is regulated at the molecular level. For example, whether post-translational modifications of PolΘ are important for its TMEJ activity and regulation is unknown.

Background

Inflammatory stress provides a clonogenic advantage towards CHIP mutant hematopoietic stem and progenitor cells (HSPCs) via different mechanisms including resistance to apoptosis, enhanced self-renewal and limited generation of reactive oxygen species. Reactive oxygen species (ROS) play a causative role in hematopoietic stem cell (HSC) dysfunction due to exit from quiescence and replication stress-induced stem cell exhaustion. Oxidative stress mediated decline in the fitness of WT hematopoietic stem cells (HSCs) may allow mutant HSCs to gain a selective advantage. Therefore, protection of WT HSCs from ROS may maintain their fitness, and prevent the selective expansion of mutant cells.

Myeloproliferative neoplasms (MPN) are heterogenous clonal hematologic neoplasms where current therapies show limited disease-modification. We previously reported that the splicing factor, SF3B1, is mutated in 5-10% of MPN correlating with myelofibrotic progression in essential thrombocythemia. This adverse phenotype contrasts with SF3B1mutationsin myelodysplasia where it is associated with milder disease. Moreover, SF3B1K666 is the dominant hotspot in MPN in contrast to K700E in MDS (abstract #185043). The mechanism by which SF3B1mutations accelerate myelofibrotic progression in JAK2V617F-mutated MPN is not understood and will be a key step towards the development of disease-modifying therapies.

Introduction:A fundamental hallmark of cancer is that tumor cells repurpose the tissue microenvironment to promote their own survival. An increased understanding of these mechanisms may lead to improved microenvironment-directed therapies, particularly in lymphoid malignancies. In classic Hodgkin lymphoma (cHL), the rare malignant Hodgkin Reed Sternberg (HRS) cells are surrounded by a CD4+ T-cell and macrophage-rich inflammatory infiltrate. Recent multiplexed immunofluorescence studies suggest that the micron-scale niche around HRS cells is composed of distinct populations of PD-L1+ macrophages and CD4+ T cells, including regulatory CTLA4+ and LAG3+ subsets (Carey et al. Blood 2017, Patel et al. Blood 2019 and Aoki et al. Cancer Discov 2020). However, the topography of the intact tumor microenvironment of cHL requires further definition. Recent single-cell RNA sequencing studies have led to important insights into the biology of cHL; however, they do not adequately capture myeloid cells, fibroblasts, and HRS cells, likely due to the relative fragility of these cells in conventional tissue dissociation protocols. In this study, we use tandem single nucleus and spatially resolved RNA sequencing to systematically dissect the pro-tumorigenic cellular niche of cHL to define potentially targetable microenvironmental dependencies.

Background: Hemophilia A is a rare X-linked bleeding disorder caused by factor VIII (FVIII) deficiency. There is a compelling need in hemophilia to better understand the causes of bleeding in females and the factors underlying FVIII inhibitor development. To meet this need we are establishing a new NHLBI funded Hemophilia A Analytical Cohort Research Program (HARP) as part of a public-private partnership to support an Intergenerational Precision Medicine Program for the study of factor VIII (FVIII) immunogenicity in severe hemophilia A (Harpf8.org).

BACKGROUND

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The Primary Immune Deficiency Treatment Consortium (PIDTC) performed a retrospective analysis of 662 patients with severe combined immunodeficiency (SCID) who received a hematopoietic cell transplantation (HCT) as first-line treatment between 1982 and 2012 in 33 North American institutions. Overall survival was higher after HCT from matched-sibling donors (MSDs). Among recipients of non-MSD HCT, multivariate analysis showed that the SCID genotype strongly influenced survival and immune reconstitution. Overall survival was similar for patients with RAG, IL2RG, or JAK3 defects and was significantly better compared with patients with ADA or DCLRE1C mutations. Patients with RAG or DCLRE1C mutations had poorer immune reconstitution than other genotypes. Although survival did not correlate with the type of conditioning regimen, recipients of reduced-intensity or myeloablative conditioning had a lower incidence of treatment failure and better T- and B-cell reconstitution, but a higher risk for graft-versus-host disease, compared with those receiving no conditioning or immunosuppression only. Infection-free status and younger age at HCT were associated with improved survival. Typical SCID, leaky SCID, and Omenn syndrome had similar outcomes. Landmark analysis identified CD4+ and CD4+CD45RA+ cell counts at 6 and 12 months post-HCT as biomarkers predictive of overall survival and long-term T-cell reconstitution. Our data emphasize the need for patient-tailored treatment strategies depending upon the underlying SCID genotype. The prognostic significance of CD4+ cell counts as early as 6 months after HCT emphasizes the importance of close follow-up of immune reconstitution to identify patients who may need additional intervention to prevent poor long-term outcome.

Hypereosinophilic syndromes (HESs) are a heterogeneous group of disorders characterized by peripheral eosinophilia and eosinophil-related end organ damage. Whereas most patients respond to glucocorticoid (GC) therapy, high doses are often necessary, and side effects are common. Dexpramipexole (KNS-760704), an orally bioavailable synthetic aminobenzothiazole, showed an excellent safety profile and was coincidentally noted to significantly decrease absolute eosinophil counts (AECs) in a phase 3 trial for amyotrophic lateral sclerosis. This proof-of-concept study was designed to evaluate dexpramipexole (150 mg orally twice daily) as a GC-sparing agent in HESs. Dual primary end points were (1) the proportion of subjects with ≥50% decrease in the minimum effective GC dose (MED) to maintain AEC <1000/μL and control clinical symptoms, and (2) the MED after 12 weeks of dexpramipexole (MEDD) as a percentage of the MED at week 0. Out of 10 subjects, 40% (95% confidence interval [CI], 12%, 74%) achieved a ≥50% reduction in MED, and the MEDD/MED ratio was significantly <100% (median, 66%; 95% CI, 6%, 98%; P = .03). All adverse events were self-limited, and none led to drug discontinuation. Affected tissue biopsy samples in 2 subjects showed normalization of pathology and depletion of eosinophils on dexpramipexole. Bone marrow biopsy samples after 12 weeks of dexpramipexole showed selective absence of mature eosinophils in responders. Dexpramipexole appears promising as a GC-sparing agent without apparent toxicity in a subset of subjects with GC-responsive HESs. Although the exact mechanism of action is unknown, preliminary data suggest that dexpramipexole may affect eosinophil maturation in the bone marrow. This study was registered at www.clinicaltrials.gov as #NCT02101138.

Mutations in FMS-like tyrosine kinase 3 (FLT3), such as internal tandem duplications (ITDs), can be found in up to 23% of patients with acute myeloid leukemia (AML) and confer a poor prognosis. Current treatment options for FLT3(ITD)-positive AMLs include genotoxic therapy and FLT3 inhibitors (FLT3i's), which are rarely curative. PARP1 inhibitors (PARP1i's) have been successfully applied to induce synthetic lethality in tumors harboring BRCA1/2 mutations and displaying homologous recombination (HR) deficiency. We show here that inhibition of FLT3(ITD) activity by the FLT3i AC220 caused downregulation of DNA repair proteins BRCA1, BRCA2, PALB2, RAD51, and LIG4, resulting in inhibition of 2 major DNA double-strand break (DSB) repair pathways, HR, and nonhomologous end-joining. PARP1i, olaparib, and BMN673 caused accumulation of lethal DSBs and cell death in AC220-treated FLT3(ITD)-positive leukemia cells, thus mimicking synthetic lethality. Moreover, the combination of FLT3i and PARP1i eliminated FLT3(ITD)-positive quiescent and proliferating leukemia stem cells, as well as leukemic progenitors, from human and mouse leukemia samples. Notably, the combination of AC220 and BMN673 significantly delayed disease onset and effectively reduced leukemia-initiating cells in an FLT3(ITD)-positive primary AML xenograft mouse model. In conclusion, we postulate that FLT3i-induced deficiencies in DSB repair pathways sensitize FLT3(ITD)-positive AML cells to synthetic lethality triggered by PARP1i's. Therefore, FLT3(ITD) could be used as a precision medicine marker for identifying AML patients that may benefit from a therapeutic regimen combining FLT3 and PARP1i's.