We have developed a cost-effective DNA methylation sequencing assay to improve monitoring of clonal hematopoiesis. By inferring cell-type proportions, this method enhances interpretation of clonal trajectories compared with interpretation based on variant allele fraction only.

CD47 is overexpressed on the surface of many types of cancer cells, including T-cell acute lymphoblastic leukemia (T-ALL) cells. In this study, we have developed a diphtheria toxin (DT)-based bivalent anti-human CD47 immunotoxin (bi-CD47-IT) for the targeted therapy of CD47+ cancers using a unique DT-resistant yeast Pichia pastoris expression system. Bi-CD47-IT demonstrated compelling in vivo efficacy in multiple T-ALL cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models. Bi-CD47-IT significantly prolonged the median survival of the tumor-bearing mice and highly effectively depleted the T-ALL blast cells in the peripheral blood, spleen, liver, bone marrow, brain, and spinal cord in the T-ALL CDX and PDX mouse models. Bi-CD47-IT cured 60% of tumor-bearing mice in a T-ALL Molt-4 CDX mouse model. Because CD47 is also expressed on normal tissues, including red blood cells and lymphocytes, specificity is a concern. We thus analyzed the in vitro binding avidity and hemagglutination of bi-CD47-IT in human red blood cells, finding no binding or hemagglutination. We further performed a toxicity study of bi-CD47-IT in humanized mice, which showed that bi-CD47-IT transiently depleted the human lymphocytes for ∼4 weeks after the 10-day treatment. No clinical adverse events were observed. As a result, bi-CD47-IT appears to possess the "optimal" binding avidity, with effective binding to human CD47+ T-ALL tumor cells, no binding to human red blood cells, and weak binding to human lymphocytes. We believe that bi-CD47-IT is a promising and safe therapeutic drug candidate for the targeted therapy of CD47+ cancers.

Immune effector cell-associated hematotoxicity (ICAHT) is a major B-cell targeted chimeric antigen receptor (CAR) T-cell related toxicity. Although ICAHT incidence and severity is documented in large B-cell lymphoma (LBCL), mantle cell lymphoma (MCL), and multiple myeloma (MM), ICAHT has not been described in B-cell acute lymphoblastic leukemia (B-ALL). Similarly, the CAR-HEMATOTOX (CAR-HT) model, designed to predict severe prolonged neutropenia (≥14 days of absolute neutrophil count [ANC] <500/μL), has been validated in LBCL, MCL, and MM, but not in B-ALL. As B-ALL bone marrow (BM) infiltration can impact cytopenias, we sought to describe ICAHT and assess CAR-HT for predicting hematotoxicity in B-ALL. In a cohort of 156 children and young adults with relapsed/refractory B-ALL, the median duration of severe neutropenia (ANC <500/μL) was 13 days (95% confidence interval, 10-16 days), with 83 (53%) experiencing grade ≥3 ICAHT. Applying CAR-HT, nearly 90% were classified as high risk, demonstrating limited discriminative power and prompting further development. Using the association identified between BM disease burden and postinfusion neutropenia (r = 0.64, P < .0001), we developed the ALL-Hematotox (ALL-HT) score, which substitutes BM disease burden for ferritin in CAR-HT. The ALL-HT score associated with severe prolonged neutropenia (area under the curve = 0.84, P < .0001), and appropriately discriminated high-risk patients (47%) who had more cumulative days of neutropenia (26 vs 4 days; P < .0001), fewer rates of complete response (88% vs 98%; P = .03), and shorter median overall survival (9.8 vs 24 months; log-rank P = .0002). ALL-HT was also validated in 2 independent cohorts. The ALL-HT score refines a widely accepted predictive model of postinfusion hematotoxicity, which is applicable in B-ALL.

Approximately 30% of patients with acute myeloid leukemia (AML) express CD7 on their myeloblasts. We have previously demonstrated that single-chain variable fragment (scFv)-based "naturally selected" CD7 chimeric antigen receptor T-cell (NS7CAR-T) therapy shows significant efficacy, with a favorable safety profile in T-cell lymphoid malignancies. Here, we derived dual variable heavy-chain domain of a heavy-chain antibody (dVHH) NS7CAR-Ts that have superior CD7 binding specificity, affinity to their scFv-based counterparts, and improved proliferative capability. In this phase 1 clinical trial, we evaluated the efficacy and safety of nanobody-based dVHH NS7CAR-Ts for patients with CD7+ refractory/relapsed AML. A cohort of 10 patients received dVHH NS7CAR-Ts across 2 dosage levels of 5 × 105/kg and 1 × 106/kg. Before enrollment, patients had undergone a median of 8 (range, 3-17) prior lines of therapy. Seven patients had prior transplants. After NS7CAR-T infusion, 7 of 10 (70%) patients achieved complete remission (CR). The median observation time was 178 days (range, 28-776). Among 7 patients who achieved CR, 3 who relapsed from prior transplants underwent a second allogeneic hematopoietic stem cell transplant (allo-HSCT). One patient remained leukemia free on day 401, and the other 2 died on day 241 and day 776, respectively, from nonrelapse-related causes. Three CR patients without consolidative (allo-HSCT) relapsed within 90 days. All the nonresponders and relapsed patients had CD7 loss. The treatment was well tolerated, with 80% experiencing mild cytokine release syndrome and none had neurotoxicity. This trial underscores the potential promising treatment of dVHH NS7CAR-Ts in providing clinical benefits with a manageable safety profile to patients with CD7+ AML, warranting further investigation. This trial was registered at www.clinicaltrials.gov as #NCT04938115.

Rising blast percentage or secondary acute myeloid leukemia (sAML) transformation in myeloproliferative neoplasms (MPNs) leads to JAK1/2 inhibitor (JAKi) therapy resistance and poor survival. Here, we demonstrate that treatment with the CDK7 inhibitor (CDK7i) SY-5609 depletes phenotypically characterized post-MPN sAML stem/progenitor cells. In cultured post-MPN sAML SET2, HEL and patient-derived (PD) post-MPN sAML cells, SY-5609 treatment inhibited growth and induced lethality while sparing normal cells. RNA-sequencing analysis after SY-5609 treatment reduced mRNA expression of MYC, MYB, CDK4/6, PIM1, and CCND1 but increased expression of CDKN1A and BCL2L1. Mass spectrometry of SY-5609-treated MPN-sAML cells also reduced c-Myc, c-Myb, PIM1, and CDK4/6 but increased p21, caspase-9, and BAD protein levels. CRISPR-mediated CDK7 depletion also reduced cell viability of HEL cells. Cytometry by time of flight (CyTOF) analysis of SY-5609-treated PD post-MPN sAML stem/progenitor cells showed reduced c-Myc, CDK6, and PU.1 but increased protein levels of CD11b, p21, and cleaved caspase-3. Cotreatment with SY-5609 and ruxolitinib was synergistically lethal in HEL, SET2, and PD post-MPN sAML cells. A CRISPR screen in sAML cells revealed BRD4, CBP, and p300 as codependencies with CDK7i. Accordingly, cotreatment with SY-5609 and the bromodomain and extra-terminal protein inhibitor (BETi) OTX015 or pelabresib or the CBP/p300 inhibitor GNE-049 was synergistically lethal in MPN-sAML cells (including those exhibiting TP53 loss). Finally, in the HEL-Luc/GFP xenograft model, compared with each agent alone, cotreatment with SY-5609 and OTX015 reduced sAML burden and improved survival without host toxicity. These findings demonstrate promising preclinical activity of CDK7i-based combinations with BETi or CBP/p300 inhibitor against advanced MPNs, including post-MPN sAML.

Red blood cell (RBC) metabolism regulates hemolysis during aging in vivo and in the blood bank. However, the genetic underpinnings of RBC metabolic heterogeneity and extravascular hemolysis at population scale are incompletely understood. On the basis of the breeding of 8 founder strains with extreme genetic diversity, the Jackson Laboratory diversity outbred population can capture the impact of genetic heterogeneity in like manner to population-based studies. RBCs from 350 outbred mice, either fresh or stored for 7 days, were tested for posttransfusion recovery, as well as metabolomics and lipidomics analyses. Metabolite and lipid quantitative trait loci (QTL) mapped >400 gene-metabolite associations, which we collated into an online interactive portal. Relevant to RBC storage, we identified a QTL hotspot on chromosome 1, mapping on the region coding for the ferrireductase 6-transmembrane epithelial antigen of the prostate 3 (Steap3), a transcriptional target to p53. Steap3 regulated posttransfusion recovery, contributing to a ferroptosis-like process of lipid peroxidation, as validated via genetic manipulation in mice. Translational validation of murine findings in humans, STEAP3 polymorphisms were associated with RBC iron content, lipid peroxidation, and in vitro hemolysis in 13 091 blood donors from the Recipient Epidemiology and Donor Evaluation Study. QTL analyses in humans identified a network of gene products (fatty acid desaturases 1 and 2, epoxide hydrolase 2, lysophosphatidylcholine acetyl-transferase 3, solute carrier family 22 member 16, glucose 6-phosphate dehydrogenase, very long chain fatty acid elongase, and phospholipase A2 group VI) associated with altered levels of oxylipins. These polymorphisms were prevalent in donors of African descent and were linked to allele frequency of hemolysis-linked polymorphisms for Steap3 or p53. These genetic variants were also associated with lower hemoglobin increments in thousands of single-unit transfusion recipients from the vein-to-vein database.

Follicular lymphoma (FL) usually requires multiple lines of therapy, and disease control remains largely insufficient with conventional chemoimmunotherapy. Several T-cell-redirecting strategies recently approved in the relapsed/refractory setting have the potential to improve outcomes and change the treatment algorithm in FL. This review focuses on the role of chimeric antigen receptor T cells and bispecific antibodies in FL, paying special attention to sequencing approaches and future directions.

Anemia is the most prevalent cytopenia in lower-risk myelodysplastic neoplasms (LR-MDS). There is a paucity of drugs for red blood cell transfusion dependence (RBC-TD), and erythropoiesis-stimulating agents (ESAs) are the mainstay of therapy in many centers. Imetelstat, an oligonucleotide telomerase inhibitor, was recently approved for adults with RBC-TD LR-MDS who are ineligible for or failed prior ESA therapy. Although not yet approved worldwide, here we spotlight the current data for imetelstat and where it may fit in the therapeutic landscape of LR-MDS.

Arterial and venous thromboses are the most significant complications in patients with myeloproliferative neoplasms (MPNs), with the primary treatment goal being thrombotic risk reduction. In MPN with no history of thrombosis, primary prevention mainly involves the use of aspirin, and cytoreduction is added in high-risk patients. However, thrombotic complications can unveil an MPN in ∼20% of cases, necessitating the initiation of both antithrombotic therapy for the thrombosis and cytoreductive treatment for the MPN. The duration of anticoagulant therapy after an initial venous thromboembolic event is subject to discussion. Furthermore, the occurrence of a thrombotic complication in patients with a known diagnosis of MPN prompts a reconsideration of both antithrombotic and hematologic management. This review uses case-based discussions to explore the management of thrombotic complications in patients with MPN. It addresses the nature and duration of antithrombotic treatments, as well as the approach to cytoreduction. Special attention is given to the place of direct oral anticoagulants and to the management of patients with MPN with splanchnic vein thrombosis, which is disproportionately common in this group.

Combining FMS-like tyrosine kinase 3 (FLT3) inhibitors with intensive chemotherapy and transplant has substantially improved acute myeloid leukemia (AML) outcomes, prompting a recent reevaluation of the historically negative prognostic effect of FLT3 internal tandem duplication. Treatment approaches may soon undergo major changes because emerging data suggest maximal intensity does not benefit all patients and measurable residual disease potentially can guide several treatment choices. Finally, recent data also suggest that FLT3 inhibitors could transform outcomes in patients unsuitable for intensive therapy. If confirmed, this has important implications for fit patients and could revolutionize the treatment paradigm.