Phase 1b trial of tagraxofusp in combination with azacitidine with or without venetoclax in acute myeloid leukemia.

ABSTRACT: CD123, a subunit of the interleukin-3 receptor, is expressed on ∼80% of acute myeloid leukemias (AMLs). Tagraxofusp (TAG), recombinant interleukin-3 fused to a truncated diphtheria toxin payload, is a first-in-class drug targeting CD123 approved for treatment of blastic plasmacytoid dendritic cell neoplasm. We previously found that AMLs with acquired resistance to TAG were re-sensitized by the DNA hypomethylating agent azacitidine (AZA) and that TAG-exposed cells became more dependent on the antiapoptotic molecule BCL-2. Here, we report a phase 1b study in 56 adults with CD123-positive AML or high-risk myelodysplastic syndrome (MDS), first combining TAG with AZA in AML/MDS, and subsequently TAG, AZA, and the BCL-2 inhibitor venetoclax (VEN) in AML. Adverse events with 3-day TAG dosing were as expected, without indication of increased toxicity of TAG or AZA+/-VEN in combination. The recommended phase 2 dose of TAG was 12 µg/kg/day for 3 days, with 7-day AZA +/- 21-day VEN. In an expansion cohort of 26 patients (median age 71) with previously untreated European LeukemiaNet adverse-risk AML (50% TP53 mutated), triplet TAG-AZA-VEN induced response in 69% (n=18/26; 39% complete remission [CR], 19% complete remission with incomplete count recovery [CRi], 12% morphologic leukemia-free state [MLFS]). Among 13 patients with TP53 mutations, 7/13 (54%) achieved CR/CRi/MLFS (CR = 4, CRi = 2, MLFS = 1). Twelve of 17 (71%) tested responders had no flow measurable residual disease. Median overall survival and progression-free survival were 14 months (95% CI, 9.5-NA) and 8.5 months (95% CI, 5.1-NA), respectively. In summary, TAG-AZA-VEN shows encouraging safety and activity in high-risk AML, including TP53-mutated disease, supporting further clinical development of TAG combinations. The study was registered on ClinicalTrials.gov as #NCT03113643.

Targeting MET and FGFR in Relapsed or Refractory Acute Myeloid Leukemia: Preclinical and Clinical Findings, and Signal Transduction Correlates.

PURPOSE: Patients with relapsed/refractory (R/R) acute myeloid leukemia (AML) have poor outcomes and require new therapies. In AML, autocrine production of hepatocyte growth factor (HGF) drives MET signaling that promotes myeloblast growth and survival, making MET an attractive therapeutic target. MET inhibition exhibits activity in AML preclinical studies, but HGF upregulation by the FGFR pathway is a common mechanism of resistance. PATIENTS AND METHODS: We performed preclinical studies followed by a Phase I trial to investigate the safety and biological activity of the MET inhibitor merestinib in combination with the FGFR inhibitor LY2874455 for patients with R/R AML. Study Cohort 1 underwent a safety lead-in to determine a tolerable dose of single-agent merestinib. In Cohort 2, dose-escalation of merestinib and LY2874455 was performed following a 3+3 design. Correlative studies were conducted. RESULTS: The primary dose-limiting toxicity (DLT) observed for merestinib alone or with LY2874455 was reversible grade 3 transaminase elevation, occurring in 2 of 16 patients. Eight patients had stable disease and one achieved complete remission (CR) without measurable residual disease. Although the MTD of combination therapy could not be determined due to drug supply discontinuation, single-agent merestinib administered at 80 mg daily was safe and biologically active. Correlative studies showed therapeutic plasma levels of merestinib, on-target attenuation of MET signaling in leukemic blood, and increased HGF expression in bone marrow aspirate samples of refractory disease. CONCLUSIONS: We provide prospective, preliminary evidence that MET and FGFR are biologically active and safely targetable pathways in AML.

Efficacy of avapritinib versus best available therapy in the treatment of advanced systemic mastocytosis.

Advanced systemic mastocytosis (AdvSM) is a rare myeloid neoplasm associated with poor overall survival (OS). This study (NCT04695431) compared clinical outcomes between patients with AdvSM treated with avapritinib in the Phase 1 EXPLORER (NCT0256198) and Phase 2 PATHFINDER (NCT03580655) trials (N = 176) and patients treated with best available therapy (BAT; N = 141). A multi-center, observational, retrospective chart review study was conducted at six study sites (four European, two American) to collect data from patients with AdvSM who received BAT; these data were pooled with data from EXPLORER and PATHFINDER. Comparisons between outcomes of OS, duration of treatment (DOT), and maximum reduction in serum tryptase were conducted between the treatment cohorts, with adjustment for key covariates. The results indicated that the avapritinib cohort had significantly better survival (adjusted hazard ratio (HR) (95% confidence interval (CI)): 0.48 (0.29, 0.79); p = 0.004) and significantly longer DOT (HR: 0.36 (0.26, 0.51); p < 0.001) compared to the BAT cohort. Additionally, the mean difference in percentage maximum reduction in serum tryptase levels was 60.3% greater in the avapritinib cohort (95% CI: -72.8, -47.9; p < 0.001). With no randomized controlled trials comparing avapritinib to BAT, these data offer crucial insights into the improved efficacy of avapritinib for the treatment of AdvSM.

BRD9 degraders as chemosensitizers in acute leukemia and multiple myeloma.

Bromodomain-containing protein 9 (BRD9), an essential component of the SWI/SNF chromatin remodeling complex termed ncBAF, has been established as a therapeutic target in a subset of sarcomas and leukemias. Here, we used novel small molecule inhibitors and degraders along with RNA interference to assess the dependency on BRD9 in the context of diverse hematological malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and multiple myeloma (MM) model systems. Following depletion of BRD9 protein, AML cells undergo terminal differentiation, whereas apoptosis was more prominent in ALL and MM. RNA-seq analysis of acute leukemia and MM cells revealed both unique and common signaling pathways affected by BRD9 degradation, with common pathways including those associated with regulation of inflammation, cell adhesion, DNA repair and cell cycle progression. Degradation of BRD9 potentiated the effects of several chemotherapeutic agents and targeted therapies against AML, ALL, and MM. Our findings support further development of therapeutic targeting of BRD9, alone or combined with other agents, as a novel strategy for acute leukemias and MM.

Practical Management of the Venetoclax-Treated Patient in Chronic Lymphocytic Leukemia and Acute Myeloid Leukemia.

Venetoclax is a potent oral, highly selective small-molecule inhibitor of the antiapoptotic B-cell lymphoma 2 protein approved for chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma in treatment-naive patients (in combination with obinutuzumab) or for patients with relapsed/refractory CLL (in combination with rituximab). Venetoclax, in combination with azacitidine, decitabine, or low-dose cytarabine, is also approved in the United States for the treatment of newly diagnosed acute myeloid leukemia (AML) in adults who are ≥ 75 years or have comorbidities that preclude use of intensive induction chemotherapy. Clinical studies of patients with CLL or AML report both hematologic (e.g., neutropenia) and nonhematologic (e.g., gastrointestinal disorders and tumor lysis syndrome) adverse events associated with administration of venetoclax. It is therefore essential to provide information on the appropriate management of venetoclax-associated side effects. This article discusses the efficacy and safety of venetoclax administration and presents strategies specifically for the management of neutropenia and certain nonhematologic adverse events in patients receiving venetoclax for the treatment of AML and CLL.

GM-CSF secreting leukemia cell vaccination for MDS/AML after allogeneic HSCT: a randomized, double-blinded, phase 2 trial.

Vaccination using irradiated, adenovirus transduced autologous myeloblasts to secrete granulocyte-macrophage colony-stimulating factor (GVAX) early after allogeneic hematopoietic stem cell transplantation (HSCT) can induce potent immune responses. We conducted a randomized phase 2 trial of GVAX after HSCT for myelodysplastic syndrome with excess blasts or relapsed/refractory acute myeloid leukemia. Myeloblasts were harvested before HSCT to generate the vaccine. Randomization to GVAX vs placebo (1:1) was stratified according to disease, transplant center, and conditioning. Graft-versus-host disease (GVHD) prophylaxis included tacrolimus and methotrexate. GVAX or placebo vaccination was started between day 30 and 45 if there was engraftment and no GVHD. Vaccines were administered subcutaneously/intradermally weekly × 3, then every 2 weeks × 3. Tacrolimus taper began after vaccine completion. A total of 123 patients were enrolled, 92 proceeded to HSCT, and 57 (GVAX, n = 30; placebo, n = 27) received at least 1 vaccination. No Common Toxicity Criteria grade 3 or worse vaccine-related adverse events were reported, but injection site reactions were more common after GVAX (10 vs 1; P = .006). With a median follow-up of 39 months (range, 9-89 months), 18-month progression-free survival, overall survival, and relapse incidence were 53% vs 55% (P = .79), 63% vs 59% (P = .86), and 30% vs 37% (P = .51) for GVAX and placebo, respectively. Nonrelapse mortality at 18 months was 17% vs 7.7% (P = .18), grade II to IV acute GVHD at 12 months was 34% vs 12% (P = .13), and chronic GVHD at 3 years was 49% vs 57% for GVAX and placebo (P = .26). Reconstitution of T, B, and natural killer cells was not decreased or enhanced by GVAX. There were no differences in serum major histocompatibility chain-related protein A/B or other immune biomarkers between GVAX and placebo. GVAX does not improve survival after HSCT for myelodysplastic syndrome/acute myeloid leukemia. This trial was registered at www.clinicaltrials.gov as #NCT01773395.

Transcriptional differences between JAK2-V617F and wild-type bone marrow cells in patients with myeloproliferative neoplasms.

The JAK2-V617F mutation is the most common cause of myeloproliferative neoplasms. Although experiments have revealed that this gain-of-function mutation is associated with myeloid blood cell expansion and increased production of white cells, red cells, and platelets, the transcriptional consequences of the JAK2-V617F mutation in different cellular compartments of the bone marrow have not yet been fully elucidated. To study the direct effects of JAK2-V617F on bone marrow cells in patients with myeloproliferative neoplasms, we performed joint single-cell RNA sequencing and JAK2 genotyping on CD34+-enriched cells from eight patients with newly diagnosed essential thrombocythemia or polycythemia vera. We found that the JAK2-V617F mutation increases the expression of interferon-response genes (e.g., HLAs) and the leptin receptor in hematopoietic progenitor cells. Furthermore, we sequenced a population of CD34- bone marrow monocytes and found that the JAK2 mutation increased expression of intermediate monocyte genes and the fibrocyte-associated surface protein SLAMF7 in these cells.

Targeting acute myeloid leukemia dependency on VCP-mediated DNA repair through a selective second-generation small-molecule inhibitor.

The development and survival of cancer cells require adaptive mechanisms to stress. Such adaptations can confer intrinsic vulnerabilities, enabling the selective targeting of cancer cells. Through a pooled in vivo short hairpin RNA (shRNA) screen, we identified the adenosine triphosphatase associated with diverse cellular activities (AAA-ATPase) valosin-containing protein (VCP) as a top stress-related vulnerability in acute myeloid leukemia (AML). We established that AML was the most responsive disease to chemical inhibition of VCP across a panel of 16 cancer types. The sensitivity to VCP inhibition of human AML cell lines, primary patient samples, and syngeneic and xenograft mouse models of AML was validated using VCP-directed shRNAs, overexpression of a dominant-negative VCP mutant, and chemical inhibition. By combining mass spectrometry-based analysis of the VCP interactome and phospho-signaling studies, we determined that VCP is important for ataxia telangiectasia mutated (ATM) kinase activation and subsequent DNA repair through homologous recombination in AML. A second-generation VCP inhibitor, CB-5339, was then developed and characterized. Efficacy and safety of CB-5339 were validated in multiple AML models, including syngeneic and patient-derived xenograft murine models. We further demonstrated that combining DNA-damaging agents, such as anthracyclines, with CB-5339 treatment synergizes to impair leukemic growth in an MLL-AF9-driven AML murine model. These studies support the clinical testing of CB-5339 as a single agent or in combination with standard-of-care DNA-damaging chemotherapy for the treatment of AML.

Midostaurin reduces relapse in FLT3-mutant acute myeloid leukemia: the Alliance CALGB 10603/RATIFY trial.

The prospective randomized, placebo-controlled CALGB 10603/RATIFY trial (Alliance) demonstrated a statistically significant overall survival benefit from the addition of midostaurin to standard frontline chemotherapy in a genotypically-defined subgroup of 717 patients with FLT3-mutant acute myeloid leukemia (AML). The risk of death was reduced by 22% on the midostaurin-containing arm. In this post hoc analysis, we analyzed the cumulative incidence of relapse (CIR) on this study and also evaluated the impact of 12 4-week cycles of maintenance therapy. CIR analyses treated relapses and AML deaths as events, deaths from other causes as competing risks, and survivors in remission were censored. CIR was improved on the midostaurin arm (HR = 0.71 (95% CI, 0.54-0.93); p = 0.01), both overall and within European LeukemiaNet 2017 risk classification subsets when post-transplant events were considered in the analysis as events. However, when transplantation was considered as a competing risk, there was overall no significant difference between the risks of relapse on the two randomized arms. Patients still in remission after consolidation with high-dose cytarabine entered the maintenance phase, continuing with either midostaurin or placebo. Analyses were inconclusive in quantifying the impact of the maintenance phase on the overall outcome. In summary, midostaurin reduces the CIR.

Reconstructing the Lineage Histories and Differentiation Trajectories of Individual Cancer Cells in Myeloproliferative Neoplasms.

Some cancers originate from a single mutation event in a single cell. Blood cancers known as myeloproliferative neoplasms (MPNs) are thought to originate when a driver mutation is acquired by a hematopoietic stem cell (HSC). However, when the mutation first occurs in individuals and how it affects the behavior of HSCs in their native context is not known. Here we quantified the effect of the JAK2-V617F mutation on the self-renewal and differentiation dynamics of HSCs in treatment-naive individuals with MPNs and reconstructed lineage histories of individual HSCs using somatic mutation patterns. We found that JAK2-V617F mutations occurred in a single HSC several decades before MPN diagnosis-at age 9 ± 2 years in a 34-year-old individual and at age 19 ± 3 years in a 63-year-old individual-and found that mutant HSCs have a selective advantage in both individuals. These results highlight the potential of harnessing somatic mutations to reconstruct cancer lineages.

Glasdegib plus low-dose cytarabine for acute myeloid leukemia: Practical considerations from advanced practitioners and pharmacists.

OBJECTIVE: Acute myeloid leukemia (AML) is primarily a disease of older adults. These patients may not be candidates for intensive treatment, and there has been an ongoing need for treatment options for this group. We review the use of glasdegib, a hedgehog-pathway inhibitor available for use in combination with low-dose cytarabine (LDAC).Data Sources: PubMed and relevant congress abstracts were searched using the term "glasdegib". In addition, based on our experience with glasdegib, we considered treatment aspects of particular relevance to pharmacists and advanced practitioners.Data Summary: In a randomized phase II study, the combination of glasdegib plus LDAC demonstrated superior overall survival versus LDAC alone (hazard ratio 0.51, 80% confidence interval 0.39-0.67, p = 0.0004). The trial reported adverse events (AEs) of special relevance for older patients, such as hematologic events, gastrointestinal toxicity, and fatigue, as well as AEs associated with Hh-pathway inhibitors (alopecia, muscle spasms, dysgeusia). Educating patients about typical AEs can facilitate adherence as well as early AE identification and proactive management. For LDAC, which is a long-established therapy in AML, various stages of delivery need consideration, with attention to individual circumstances. Practical measures such as dispensing a longer supply can reduce the number of return clinic visits, providing a meaningful difference for many patients. CONCLUSIONS: Pharmacists and advanced practitioners play important roles in treatment with glasdegib plus LDAC. Ultimately, framing plans for treatment delivery within the individual circumstances of each patient may enable them to stay on therapy longer, giving them the greatest potential to achieve benefit.

Midostaurin: Nursing Perspectives on Managing Treatment and Adverse Events in Patients With FLT3 Mutation-Positive Acute Myeloid Leukemia and Advanced Systemic Mastocytosis.

BACKGROUND: Acute myeloid leukemia (AML) and advanced systemic mastocytosis (SM) are clonal diseases of the blood. Prognoses for patients with FMS-like tyrosine kinase 3 (FLT3) mutation-positive AML and those with advanced SM are poor. In the United States, midostaurin was approved in 2017 in combination with standard chemotherapy in patients with newly diagnosed FLT3 mutation-positive AML and as a single agent in patients with advanced SM. OBJECTIVES: This article aims to improve oncology nurses' knowledge about the benefits and risks of midostaurin therapy and to provide guidance on the identification and management of eligible patients. METHODS: The clinical data that supported the U.S. Food and Drug Administration's approval of midostaurin are reviewed, and supporting safety and management considerations are provided based on the authors' experiences as nurses and advanced practice providers caring for patients who received midostaurin during these key clinical trials. FINDINGS: Nausea and vomiting are the most frequent nonhematologic adverse events reported with midostaurin; therefore, administer midostaurin with antiemetics, and recommend taking it with food. Care should be taken when midostaurin is coadministered with strong CYP3A4 inhibitors.

Vulnerabilities in mIDH2 AML confer sensitivity to APL-like targeted combination therapy.

Although targeted therapies have proven effective and even curative in human leukaemia, resistance often ensues. IDH enzymes are mutated in ~20% of human AML, with targeted therapies under clinical evaluation. We here characterize leukaemia evolution from mutant IDH2 (mIDH2)-dependence to independence identifying key targetable vulnerabilities of mIDH2 leukaemia that are retained during evolution and progression from early to late stages. Mechanistically, we find that mIDH2 leukaemia are metastable and vulnerable at two distinct levels. On the one hand, they are characterized by oxidative and genotoxic stress, in spite of increased 1-carbon metabolism and glutathione levels. On the other hand, mIDH2 leukaemia display inhibition of LSD1 and a resulting transcriptional signature of all-trans retinoic acid (ATRA) sensitization, in spite of a state of suppressed ATRA signalling due to increased levels of PIN1. We further identify GSH/ROS and PIN1/LSD1 as critical nodes for leukaemia maintenance and the combination of ATRA and arsenic trioxide (ATO) as a key therapeutic modality to target these vulnerabilities. Strikingly, we demonstrate that the combination of ATRA and ATO proves to be a powerfully synergistic and effective therapy in a number of mouse and human mIDH1/2 leukemic models. Thus, our findings pave the way towards the treatment of a sizable fraction of human AMLs through targeted APL-like combinatorial therapies.

Single-Cell RNA-Seq Reveals AML Hierarchies Relevant to Disease Progression and Immunity.

Acute myeloid leukemia (AML) is a heterogeneous disease that resides within a complex microenvironment, complicating efforts to understand how different cell types contribute to disease progression. We combined single-cell RNA sequencing and genotyping to profile 38,410 cells from 40 bone marrow aspirates, including 16 AML patients and five healthy donors. We then applied a machine learning classifier to distinguish a spectrum of malignant cell types whose abundances varied between patients and between subclones in the same tumor. Cell type compositions correlated with prototypic genetic lesions, including an association of FLT3-ITD with abundant progenitor-like cells. Primitive AML cells exhibited dysregulated transcriptional programs with co-expression of stemness and myeloid priming genes and had prognostic significance. Differentiated monocyte-like AML cells expressed diverse immunomodulatory genes and suppressed T cell activity in vitro. In conclusion, we provide single-cell technologies and an atlas of AML cell states, regulators, and markers with implications for precision medicine and immune therapies. VIDEO ABSTRACT.

Phase I Trial of Autologous CAR T Cells Targeting NKG2D Ligands in Patients with AML/MDS and Multiple Myeloma.

NKG2D ligands are widely expressed in solid and hematologic malignancies but absent or poorly expressed on healthy tissues. We conducted a phase I dose-escalation study to evaluate the safety and feasibility of a single infusion of NKG2D-chimeric antigen receptor (CAR) T cells, without lymphodepleting conditioning in subjects with acute myeloid leukemia/myelodysplastic syndrome or relapsed/refractory multiple myeloma. Autologous T cells were transfected with a γ-retroviral vector encoding a CAR fusing human NKG2D with the CD3ζ signaling domain. Four dose levels (1 × 106-3 × 107 total viable T cells) were evaluated. Twelve subjects were infused [7 acute myeloid leukemia (AML) and 5 multiple myeloma]. NKG2D-CAR products demonstrated a median 75% vector-driven NKG2D expression on CD3+ T cells. No dose-limiting toxicities, cytokine release syndrome, or CAR T cell-related neurotoxicity was observed. No significant autoimmune reactions were noted, and none of the ≥ grade 3 adverse events were attributable to NKG2D-CAR T cells. At the single injection of low cell doses used in this trial, no objective tumor responses were observed. However, hematologic parameters transiently improved in one subject with AML at the highest dose, and cases of disease stability without further therapy or on subsequent treatments were noted. At 24 hours, the cytokine RANTES increased a median of 1.9-fold among all subjects and 5.8-fold among six AML patients. Consistent with preclinical studies, NKG2D-CAR T cell-expansion and persistence were limited. Manufactured NKG2D-CAR T cells exhibited functional activity against autologous tumor cells in vitro, but modifications to enhance CAR T-cell expansion and target density may be needed to boost clinical activity.

Isocitrate dehydrogenase 1 and 2 mutations, 2-hydroxyglutarate levels, and response to standard chemotherapy for patients with newly diagnosed acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) cells harboring mutations in isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) produce the oncometabolite 2-hydroxyglutarate (2HG). This study prospectively evaluated the 2HG levels, IDH1/2 mutational status, and outcomes of patients receiving standard chemotherapy for newly diagnosed AML. METHODS: Serial samples of serum, urine, and bone marrow aspirates were collected from patients newly diagnosed with AML, and 2HG levels were measured with mass spectrometry. Patients with baseline serum 2HG levels greater than 1000 ng/mL or marrow pellet 2HG levels greater than 1000 ng/2 × 106 cells, which suggested the presence of an IDH1/2 mutation, underwent serial testing. IDH1/2 mutations and estimated variant allele frequencies were identified. AML characteristics were compared with the Wilcoxon test and Fisher's exact test. Disease-free survival and overall survival (OS) were evaluated with log-rank tests and Cox regression. RESULTS: Two hundred and two patients were treated for AML; 51 harbored IDH1/2 mutations. IDH1/2-mutated patients had significantly higher 2HG levels in serum, urine, bone marrow aspirates, and aspirate cell pellets than wild-type patients. A serum 2HG level greater than 534.5 ng/mL was 98.8% specific for the presence of an IDH1/2 mutation. Patients with IDH1/2-mutated AML treated with 7+3-based induction had a 2-year event-free survival (EFS) rate of 44% and a 2-year OS rate of 57%. There was no difference in complete remission rates, EFS, or OS between IDH1/2-mutated and wild-type patients. Decreased serum 2HG levels on day 14 as a proportion of the baseline were significantly associated with improvements in EFS (P = .047) and OS (P = .019) in a multivariate analysis. CONCLUSIONS: Among patients with IDH1/2-mutated AML, 2HG levels are highly specific for the mutational status at diagnosis, and they have prognostic relevance in patients receiving standard chemotherapy.

Neuropathology of a Case With Fatal CAR T-Cell-Associated Cerebral Edema.

Chimeric antigen receptor (CAR) T cells are a new and powerful class of cancer immunotherapeutics that have shown potential for the treatment of hematopoietic malignancies. The tremendous promise of this approach is tempered by safety concerns, including potentially fatal neurotoxicity, sometimes but not universally associated with cytokine release syndrome. We describe the postmortem examination of a brain from a 21-year-old patient with relapsed pre-B cell acute lymphoblastic leukemia (ALL) who died from fulminant cerebral edema following CAR T-cell infusion. We found a range of changes that included activation of microglia, expansion of perivascular spaces by proteinaceous exudate, and clasmatodendrosis-a beading of glial fibrillary acidic protein consistent with astrocyte injury. Notably, within the brain parenchyma, we identified only infrequent T cells and did not identify ALL cells or CAR T cells. The overall findings are nonspecific but raise the possibility of astrocyte and blood-brain barrier dysfunction as a potential etiology of fatal CAR T-cell neurotoxicity in this patient.

Increased neutrophil extracellular trap formation promotes thrombosis in myeloproliferative neoplasms.

Thrombosis is a major cause of morbidity and mortality in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), clonal disorders of hematopoiesis characterized by activated Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling. Neutrophil extracellular trap (NET) formation, a component of innate immunity, has been linked to thrombosis. We demonstrate that neutrophils from patients with MPNs are primed for NET formation, an effect blunted by pharmacological inhibition of JAK signaling. Mice with conditional knock-in of Jak2V617F, the most common molecular driver of MPN, have an increased propensity for NET formation and thrombosis. Inhibition of JAK-STAT signaling with the clinically available JAK2 inhibitor ruxolitinib abrogated NET formation and reduced thrombosis in a deep vein stenosis murine model. We further show that expression of PAD4, a protein required for NET formation, is increased in JAK2V617F-expressing neutrophils and that PAD4 is required for Jak2V617F-driven NET formation and thrombosis in vivo. Finally, in a population study of more than 10,000 individuals without a known myeloid disorder, JAK2V617F-positive clonal hematopoiesis was associated with an increased incidence of thrombosis. In aggregate, our results link JAK2V617F expression to NET formation and thrombosis and suggest that JAK2 inhibition may reduce thrombosis in MPNs through cell-intrinsic effects on neutrophil function.

Exploiting an Asp-Glu "switch" in glycogen synthase kinase 3 to design paralog-selective inhibitors for use in acute myeloid leukemia.

Glycogen synthase kinase 3 (GSK3), a key regulatory kinase in the wingless-type MMTV integration site family (WNT) pathway, is a therapeutic target of interest in many diseases. Although dual GSK3α/ß inhibitors have entered clinical trials, none has successfully translated to clinical application. Mechanism-based toxicities, driven in part by the inhibition of both GSK3 paralogs and subsequent ß-catenin stabilization, are a concern in the translation of this target class because mutations and overexpression of ß-catenin are associated with many cancers. Knockdown of GSK3α or GSK3ß individually does not increase ß-catenin and offers a conceptual resolution to targeting GSK3: paralog-selective inhibition. However, inadequate chemical tools exist. The design of selective adenosine triphosphate (ATP)-competitive inhibitors poses a drug discovery challenge due to the high homology (95% identity and 100% similarity) in this binding domain. Taking advantage of an Asp133→Glu196 "switch" in their kinase hinge, we present a rational design strategy toward the discovery of paralog-selective GSK3 inhibitors. These GSK3α- and GSK3ß-selective inhibitors provide insights into GSK3 targeting in acute myeloid leukemia (AML), where GSK3α was identified as a therapeutic target using genetic approaches. The GSK3α-selective compound BRD0705 inhibits kinase function and does not stabilize ß-catenin, mitigating potential neoplastic concerns. BRD0705 induces myeloid differentiation and impairs colony formation in AML cells, with no apparent effect on normal hematopoietic cells. Moreover, BRD0705 impairs leukemia initiation and prolongs survival in AML mouse models. These studies demonstrate feasibility of paralog-selective GSK3α inhibition, offering a promising therapeutic approach in AML.