Current therapies, including autologous chimeric antigen receptor (CAR) T-cell immunotherapy, fail to cure half of infants with KMT2A-rearranged acute lymphoblastic leukemia (KMT2Ar-ALL), a disease characterized by frequent central nervous system involvement, poor treatment response, early relapse, and lineage switching. More effective treatment strategies, including the availability of off-the-shelf immunotherapies, is particularly relevant in infants. PROM1/CD133 is a direct target of KMT2A-fusion oncoproteins and is expressed on leukemic cells. Allogeneic invariant natural killer T (iNKT) cells, "innately" more powerful effectors than T cells, can be deployed off-the-shelf without risk of acute graft-versus-host disease. Here, we equip iNKT cells with CD19- and/or CD133-targeting CARs, and investigate their antileukemia activity against KMT2Ar-ALL in relevant in vitro and in vivo models. Compared with monospecific counterparts and dual, bispecific CAR T cells, bispecific CD19-CD133 CAR-iNKT cells have a more potent antileukemia activity, effectively targeting both CAR antigen-high and -low leukemia. Bispecific CAR-iNKT cells eradicate medullary and, notably, leptomeningeal leukemia, and induce sustained remissions without discernible hematologic toxicity. Mechanistically, the more potent antileukemia effect of CAR-iNKT cells over CAR T cells is mediated by a pronounced CAR-dependent and CAR antigen-dependent upregulation of the innate activating receptor NKG2D on CAR-iNKT cells, and its engagement by its corresponding ligands on KMT2Ar-ALL cells. This ensures effective leukemia targeting even with downregulation of CD133 or CD19. Thus, by engaging with 2 different types of leukemia-associated antigens, that is, CAR antigens and NKG2D ligands, CAR-iNKT cells provide a powerful platform for the treatment of KMT2Ar-ALL. This approach can be readily adapted for other high-risk malignancies, including those with otherwise difficult to target leptomeningeal involvement.

The acquisition of N-glycosylation sites that are occupied by oligomannose-type glycans in the immunoglobulin complementarity-determining region (CDR) is an early, clonal, tumor-specific identifier of follicular lymphoma (FL). CDR-located N-glycosylation sites are also acquired in germinal center B-cell-like diffuse large B-cell lymphomas (GCB-DLBCLs), but their significance is less defined. We used RNA sequencing immunoglobulin assembly to determine frequency and CDR location of the acquired N-glycosylation sites (AGSs) in 2 independent DLBCL cohorts. Composition of the glycans occupying the AGSs was determined using liquid chromatography-mass spectrometry and correlated with cell of origin, FL signature (defined by EZB phenotype or BCL2 translocation), transcript profile, and clinical outcome. CDR-located AGSs were observed in 41% to 46% of GCB-DLBCLs but were rare in other DLBCLs. Only CDR-located AGSs of DLBCL with an FL signature were occupied by oligomannose-type glycans. These DLBCLs were termed Mann-type DLBCL. Conversely, the AGSs of the other DLBCLs were either nonglycosylated or occupied by complex-type glycans. Mann-type status was an independent marker of short progression-free survival and overall survival. In contrast, the other GCB-DLBCLs, including those with an FL signature but without AGSs, had the best outcomes. Mann-type DLBCLs overexpressed gene sets of cell growth, survival, and cycling, and underexpressed proinflammatory and apoptotic pathways, irrespective of the presence of concomitant MYC translocations. The acquisition of Mann-type glycans is a highly selective environmental pressure enabling the identification of an aggressive GCB-DLBCL type with origin related to FL. The detection of AGSs in the CDR of GCB-DLBCLs with an FL signature defines Mann-type DLBCLs, refines prognosis, and marks a precise tumor interaction to block early therapeutically.

Genomic profiling in patients with chronic-phase chronic myeloid leukemia (CP-CML) demonstrated somatic variants in blood cancer-related gene variants (CGVs) and rearrangements associated with the formation of the Philadelphia chromosome (Ph-associated rearrangements) at diagnosis, collectively termed additional genetic abnormalities (AGAs). AGAs had a negative impact on failure-free survival (FFS) and molecular response in imatinib-treated patients. We investigated whether treatment with more potent therapies could overcome the negative impact of AGAs at diagnosis. Targeted RNA-based next-generation sequencing was performed on diagnostic samples of 315 patients consecutively enrolled in 4 clinical trials of frontline potent tyrosine kinase inhibitors (TKIs) in CP-CML. AGAs were present in 34% of patients at diagnosis, including 20% harboring CGVs and 18% with Ph-associated rearrangements (4% had both). Although the negative impact of Ph-associated rearrangements was overcome by more potent inhibitors, patients with CGVs continued to experience inferior outcomes. This result was largely attributable to patients with ASXL1 variants, observed in 7% overall. Patients harboring ASXL1 variants also had inferior outcomes compared with those with wild-type ASXL1 in terms of 12-month major molecular response (55% vs 83%; P = .001), 2-year FFS (61% vs 91%; P < .001), and notably, the development of treatment-emergent BCR::ABL1 kinase domain mutations at 2 years (35% vs 1%; P < .001). In multivariable models, both CGVs and ASXL1 variants were predictors of each outcome. Treatment with frontline potent TKIs overcame the negative impact of Ph-associated rearrangements observed with frontline imatinib. However, inferior outcomes were still associated with the presence of CGVs. The acquisition of TKI-resistant BCR::ABL1 mutations was almost exclusively associated with mutated ASXL1 at diagnosis.

Histone deacetylase inhibitors (HDACis) are valued treatment options for patients with T-cell malignancies. Romidepsin is a selective class I HDACi initially approved for patients with relapsed or refractory cutaneous and peripheral T-cell lymphomas (PTCLs). Romidepsin was withdrawn from its PTCL indication following a negative randomized phase 4 study (romidepsin-CHOP [cyclophosphamide, doxorubicin hydrochloride (hydroxydaunorubicin), vincristine sulfate (Oncovin), and prednisone]) that showed no benefit over CHOP alone, further diminishing options for patients. Herein, we describe the development of, to our knowledge, a first-in-class polymer nanoparticle (PNP) of romidepsin using an innovative amphiphilic diblock copolymer-based nanochemistry platform. Nanoromidepsin exhibited superior pharmacologic properties with improved tolerability and safety in murine models of T-cell lymphoma (TCL). The PNP also exhibited superior antitumor efficacy in multiple models, including in vitro TCL cell lines, ex vivo samples from patients with large granular lymphocyte (LGL) leukemia, and murine TCL xenografts. Nanoromidepsin demonstrated greater accumulation in tumors and a statistically significant improvement in overall survival compared with romidepsin in murine xenograft models. These findings justify the clinical development of nanoromidepsin in patients with T-cell malignancies.

Success of chimeric antigen receptor (CAR) T-cell therapy in lymphoid malignancies has not yet been recapitulated in acute myeloid leukemia (AML). We developed CAR T cells targeting CD371 with a mutated CD28 costimulatory domain to limit T-cell exhaustion, and constitutive interleukin-18 (IL-18) secretion to enhance immune function (CD371/SAVVY/IL-18 CAR). We initiated a phase 1 trial (NCT06017258), successfully manufactured and administered CD371/SAVVY/IL-18 CAR T cells in 5 patients with relapsed/refractory AML and observed expansion following a single infusion of 3 × 104 or 3 × 105 CAR T cells per kg; 3 patients refractory to ≥5 lines of therapy and postallogeneic transplant exhibited AML clearance and no evidence of graft-versus-host disease. Dose-limiting toxicity in the 2 patients treated with 3 × 105 CAR T cells per kg dose (prolonged cytopenias with marrow hypoplasia; severe cytokine release syndrome) led to dose reduction to 3 × 104 CAR T cells per kg in the following 3 patients. Single-cell analyses revealed that circulating CAR T cells in responders included predominantly cytotoxic CD8+ effector T cells 2 weeks after infusion while coexisting natural killer (NK) cells expressed markers of activation. This pilot study highlights the activity of low-dose IL-18 "armored" CAR T cells against refractory AML and their potential to promote CAR T-cell cytotoxicity and innate endogenous antitumor immunity. This trial was registered at www.ClinicalTrials.gov as #NCT06017258.

Congenital thrombotic thrombocytopenic purpura (cTTP) is an ultrarare thrombotic microangiopathy mediated through inherited deficiency in a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13 (ADAMTS13). To date, >200 ADAMTS13 genetic variants have been associated with cTTP. We report longitudinal follow-up from the UK TTP registry in 104 confirmed cTTP cases (91 consented for follow-up) in a large multiethnic national cohort of patients with cTTP, including a large Black African cTTP cohort. A total of 71 ADAMTS13 variants were identified, with N-terminal variants associated with earlier age at presentation. During the follow-up period (median, 63 months; range, 1-179), 80.2% of patients received regular (plasma derived) prophylaxis, which reduced end organ damage, including stroke/transient ischemic attack (19.0%-1.5%) and renal impairment during follow-up. Postpresentation acute TTP episodes were reduced with prophylaxis (0.68 vs 0.06 acute episodes per follow-up year). Despite regular prophylaxis, symptom control remained apparent on plasma-derived therapy (including headache 42.6%, depression/anxiety 13.2%, fatigue 16.2%, and abdominal pain 13.2%). Most patients with cTTP in the United Kingdom have now switched to recombinant ADAMTS13 (n = 43 [58.9%]), owing to inadequate symptom control (53.5%), plasma reactions (30.2%), or subclinical disease activity (16.3%). This work shows the breadth of ADAMTS13 genetic variants in cTTP and demonstrates efficacy of regular prophylaxis in (1) reducing acute TTP episodes and (2) preventing end organ damage, but despite advances, cTTP related symptoms and the use of blood products remained problematic.

Venetoclax-obinutuzumab (Ven-Obi) is a standard first-line therapy for chronic lymphocytic leukemia (CLL). The impact of age, fitness, and dose reductions remains unclear. We analyzed patients treated with Ven-Obi in the CLL13 and CLL14 trials, excluding patients with TP53 aberrations. Fitness was assessed using the cumulative illness rating scale (CIRS, >6) and creatinine clearance (≤70 mL/min). Among 410 patients (median age, 67 years), 55.7% were unfit (median age, 72 years) and 44.3% were fit (median age, 58 years). Overall response rate (ORR) was 89.5% in unfit and 96.1% in fit patients. Rates of undetectable minimal residual disease (uMRD) at <10-4 were 80.3% in unfit and 85.1% in fit patients. Progression-free survival (PFS) at 3 years was 86.4% vs 87.5% (hazard ratio, [HR], 1.12; 95% confidence interval [CI], 0.70-1.81; P = .63). Overall survival at 3 years was 91.8% vs 96.9% (HR, 2.02; 95% CI, 0.90-4.55; P = .088). Adverse events included neutropenia (62.7% unfit, 56.9% fit), infusion-related-reactions (44.3% unfit, 56.9% fit), fatigue (15.8% unfit, 35.9% fit), and infections (57.5% unfit, 69.6% fit). Venetoclax dose reductions of <80% occurred in 39.6% of unfit and 17.6% of fit patients, with lower ORR (83.3% vs 98.2%) and uMRD rates (74.2% vs 87.9%) in those with reduced dose intensities, but similar PFS. Dose reductions of <70% were associated with shorter PFS. Overall, this study shows comparable efficacy and toxicity of Ven-Obi in fit and unfit patients with CLL.

Bispecific T-cell engagers (TCEs) targeting B-cell maturation antigen (BCMA) and CD3, induce deep hematologic responses in ∼60% of heavily pretreated patients with multiple myeloma (MM). We and others found that high tumor burden leads to resistance to TCE and novel strategies are urgently needed to improve responses in this setting. Ikaros degraders, including immunomodulatory drugs (IMiDs) and cereblon E3 ligase modulatory drugs (CELMoDs), represent logical partners for TCEs due to their direct anti-MM effects and additional immune-stimulatory activity; however, it is unclear how to optimally combine them with TCEs. Taking advantage of the immunocompetent IMiD-sensitive Vk∗MYChCRBN murine model of MM, we optimized strategies to overcome primary resistance to BCMA-TCEs and achieve sustained remission, while maintaining a manageable safety profile. The addition of anti-programmed cell death protein 1 (PD1) and pomalidomide reduced the T-cell exhaustion that occurs in response to TCEs in high tumor burden settings. This allowed for a higher degree of T-cell activation and significant improvement in response rates but also increased risk of lethal cytokine release syndrome (CRS). To moderate the response and prevent CRS, we evaluated Ikaros degraders and dexamethasone (DEX) with step-up-dosed TCEs. Pretreatment with iberdomide and DEX reshaped the bone marrow T-cell compartment, promoted infiltration of naïve T cells, and generated 100% response rates and the longest survival in subjects with high tumor burden. This was accompanied by more favorable T-cell profiling, with limited expansion of regulatory T cells and exhaustion. Overall, administering a TCE after DEX and iberdomide treatments provided deeper and more durable responses with a reduced risk of CRS.

Obesity is a major health issue and a risk factor for venous thromboembolic disease. Plasminogen activator inhibitor 1 (PAI-1), encoded by the gene SERPINE1, is a negative regulator of fibrinolysis and has been associated with obesity. The liver, which senses obesity-induced metabolic stress, is a key determinant of circulating PAI-1 levels. However, the mechanisms underlying the increased PAI-1 expression in obesity are unclear. This study investigated the upstream regulation of PAI-1 and its role in fibrinolysis and deep vein thrombosis (DVT). Compared with lean mice, diet-induced obesity mice presented significantly shorter fibrinolysis times and larger venous thrombi, largely due to increased hepatocyte expression of PAI-1. A publicly available single-cell RNA sequence data set from the livers of individuals with obesity suggested that increased PAI-1 expression may be related to reduced hepatocyte farnesoid X receptor (FXR) signaling. FXR activation also suppressed Serpine1 mRNA and PAI-1 protein expression levels in both mice and primary mouse hepatocytes (MPHs), but a decrease in PAI-1 in MPHs of Fxr-null mice after FXR activation was not observed. Both Fxr-null mice and Fxrfl/fl mice with AAV8-TBG-Cre exhibited significantly elevated plasma PAI-1, resulting in further impaired fibrinolysis and increased DVT burden. Dual-luciferase reporter assays and chromatin immunoprecipitation suggested that FXR activation directly represses Serpine1 transcription. Importantly, tropifexor treatment of obese mice lowered plasma PAI-1 levels and further alleviated fibrinolysis and the DVT load. These findings suggest that targeting FXR in hepatocytes may improve fibrinolysis and reduce DVT risk.

Acute myeloid leukemia (AML) is characterized by a low 5-year survival rate. Despite having many clinical metrics to assess patient prognosis, there remain opportunities to improve risk stratification. We hypothesized that an underexplored resource to examine the prognosis of patients with AML is plasma metabolome. Circulating metabolites are influenced by patients' clinical status and can serve as accessible cancer biomarkers. To establish a resource of circulating metabolites in genetically diverse patients with AML, we performed an unbiased metabolomic and lipidomic analysis of 231 diagnostic AML plasma samples before treatment with intensive chemotherapy. Intriguingly, circulating metabolites were highly associated with the mutation status within the AML cells. Furthermore, lipids were associated with refractory status. We established a machine learning algorithm trained on chemotherapy-refractory-associated lipids to predict patient survival. Cox regression and Kaplan-Meier analysis demonstrated that the high-risk lipid signature predicted overall survival in this patient cohort. Impressively, the top lipid in the high-risk lipid signature, sphingomyelin (d44:1), was sufficient to predict overall survival in both the original data set and an independent validation data set. Overall, this research underscores the potential of circulating metabolites to capture AML heterogeneity and lipids to be used as potential AML biomarkers.

The discovery of calreticulin (CALR) mutations in patients with myeloproliferative neoplasms (MPNs) has paved the way for the elucidation of a unique disease mechanism that is particularly well suited to targeting by biologics. All MPN-associated pathogenic CALR mutations are characterized by a frameshift, resulting in translation of the same neoantigen peptide. This neoantigen directly activates the thrombopoietin receptor, leading to uncontrolled neoplastic cell proliferation. Current therapeutic approaches for MPNs are focused primarily on blood count control. Furthermore, current approaches are neither disease modifying nor clonally selective. However, because the mutant CALR neoantigen peptide is functional and not expressed in normal physiology, it is an ideal drug target. Here, we review the structure and function of mutant CALR, including the subtle yet clinically and therapeutically relevant differences between the 2 most commonly occurring types of mutation. We also review the current therapeutic landscape for CALR-mutated MPNs, highlighting the areas in which current approaches are inadequate. Finally, we review ongoing clinical and preclinical experimental approaches for targeting mutant CALR in MPNs in a clonally selective manner using monoclonal antibodies, bispecific antibodies, cancer vaccination, chimeric antigen receptor T cells, and antibody-drug conjugates. Taken together, we expect that ongoing developments in mutant CALR-targeted therapeutics will lead to promising novel strategies for long-term disease control.