Immunoglobulin light chain (AL) amyloidosis is a plasma cell disorder characterized by progressive organ dysfunction secondary to deposition of organized immunoglobulin light chain aggregates. Achievement of rapid and deep normalization of involved immunoglobulin free light chains is necessary to maximize chances of reversibility of organ dysfunction, which in turn results in improved quality and length of life. There are no FDA-approved therapies for patients with relapsed and/or refractory immunoglobulin light chain (AL) amyloidosis. B cell maturation antigen (BCMA)-targeting bispecific T cell engagers teclistimab and elranatamab have shown high activity and acceptable safety profile in relapsed and/or refractory multiple myeloma patients, leading to their FDA approval. Herein we report on safety and efficacy of elranatamab for patients with relapsed and/or refractory AL amyloidosis. We treated 9 consecutive, advanced-stage AL amyloidosis patients with Elranatamab single agent, observing a 100% overall response and 67% complete response rate, including minimal residual disease (MRD)-negativity, with expected toxicities. Median time to hematological response was 9 days (6-24), with deep suppression in involved free light chains observed within one cycle of therapy, translating in cardiac and renal responses at 3-6 months. These data support prospective studies exploring Elranatamab in relapsed and/or refractory AL amyloidosis patients.

Clinical resistance or intolerance to tyrosine kinase inhibitors (TKIs) remains challenging for the treatment of chronic myeloid leukemia (CML) and Philadelphia-chromosome positive acute lymphoblastic leukemia (Ph+ ALL) with central nervous system (CNS) relapse. Therapeutic options are currently limited for patients who developed the gatekeeper mutations or compound mutations. Here, we describe the preclinical profile of TGRX-678, an allosteric inhibitor designed to Specifically Target the ABL1 Myristoyl Pocket (STAMP), with potent anti-proliferative activity against the majority of ATP site mutants of BCR::ABL1 and minimal off-target cytotoxicity. When combined with ponatinib, TGRX-678 synergistically re-sensitizes the highly resistant compound mutants and T315M to growth inhibition at clinically achievable concentrations. TGRX-678 exhibits relatively high cell permeability and is not a substrate of drug efflux transporters, namely ABCB1 and ABCG2. It also demonstrates a markedly improved in vivo pharmacokinetic (PK) profile and higher oral bioavailability compared to asciminib. Importantly, TGRX-678 penetrates the blood-brain barrier (BBB) and exhibits in vivo efficacy in a murine CNS blast crisis leukemia model. Collectively, these findings suggest that TGRX-678 is a novel BCR::ABL1 allosteric inhibitor with high selectivity, potency and unique pharmacologic features, which has the potential to treat relapse or refractory CML and Ph+ ALL, even with CNS involvement.

Leukemias with NUP98 rearrangements exhibit heterogeneous phenotypes such as acute myeloid leukemia (AML), T-acute lymphoblastic leukemia (T-ALL), or myelodysplastic syndrome/neoplasms (MDS) associated with fusion partners, whereas the mechanism responsible for this heterogeneity is poorly understood. Through genome-wide mutational and transcriptional analyses of 177 NUP98-rearranged leukemias, we show that cooperating alterations are associated with differentiation status even among leukemias sharing the same NUP98 fusions, such as NUP98::KDM5A acute megakaryocytic leukemia (AMKL) with RB1 loss or T-ALL with NOTCH1 mutations. CUT&RUN profiling of in vitro cord blood CD34+ cell (cbCD34) models of major NUP98 fusions revealed that NUP98 fusion oncoproteins directly regulate differentiation-related genes contributing to the disease phenotypes, represented by NUP98::KDM5A binding to MEIS2 or GFI1B for megakaryocyte differentiation. In patient samples, NUP98-fusion oncoprotein binding patterns are heterogeneous, potentially shaped by somatic mutations and differentiation status. Using cbCD34 models and CRISPR/Cas9 gene editing, we show that RB1 loss cooperates with NUP98::KDM5A by blocking terminal differentiation toward platelets and expanding megakaryocyte-like cells, whereas WT1 frameshift mutations skew differentiation toward dormant lymphoid-myeloid primed progenitor cells and cycling granulocyte-monocyte progenitor cells, providing evidence for NUP98-rearranged leukemia phenotypes affected by cooperating alterations. NUP98::KDM5A cbCD34 models with RB1 or WT1 alterations have different sensitivities to menin inhibition, suggesting that cellular differentiation provides stage-specific menin dependencies and resistance mechanisms that can be leveraged for future treatment strategies for NUP98-rearranged leukemia.

Targeting metabolic dependencies and "starving" malignant cells have long been considered as potential strategies to treat cancer. However, with rare exceptions, the implementation of these maneuvers has been fraught with limited activity and lack of specificity. Multiple cytoplasmic and mitochondrial transaminases catalyze reactions that lead to amino acid catabolism. These enzymes use alpha-ketoglutarate (αKG) as a nitrogen acceptor, and accumulation of the competitive inhibitor metabolite D-2-HG perturbs their function. We postulated that exogenous αKG supplementation would influence the directionality of these reactions and deplete amino acids in cancer cells. Using B cell lymphoma as a model system, we found that αKG mediates a rapid and sustained amino acid depletion, principally of aspartate and branched-chain leucine, valine and isoleucine. The decrease in leucine levels influenced MTORC1 sub-cellular movement, suppressed its activity and associated with inhibition of B cell lymphoma growth in vitro and in vivo Increasing import of aspartate or leucine levels in the lymphoma cells, genetically forcing MTORC1 lysosomal localization or blocking leucine catabolism through BCAT2 deletion, all blunted the anti-lymphoma effects of αKG. In addition, long term dietary supplementation of αKG, a toxicity free strategy, significantly hindered lymphoma development in Eµ-Myc mice, in association with amino acid perturbation and impaired energy generation. We posit that αKG supplementation, which has been shown to improve health and lifespan in mice, also encodes marked anti-cancer properties.

During thrombopoiesis, megakaryocytes (MKs) transform their cytoplasm into proplatelets through complex cytoskeletal rearrangements. The shear force of blood flow releases newly formed platelets from the proplatelets into the bloodstream. Defects at any phase of this process can impair platelet production. While various non-coding RNAs have been identified as regulators of platelet production, the regulatory mechanisms of thrombopoiesis remain to be further investigated. Despite the high abundance of circular RNAs (circRNAs) in platelets, their role in platelet production is unclear. In this study, using RNA-seq and bioinformatics analysis, we identified circFUT8 as a novel circRNA that increases as hematopoietic stem cells from human umbilical cord blood differentiate into mature MKs, showing high expression in these mature cells. Knockdown of circFUT8 led to diminished proplatelet formation (PPF) and abnormal demarcation membrane system (DMS) formation in human cultured MKs. Additionally, inhibition of circFut8 in vivo decreased murine platelet counts. CircFut8 deficiency reduced the number of MKs in contact with sinusoids. Mechanistically, we revealed that circFUT8 interacts with insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) to stabilize tensin 1 (TNS1) mRNA in an m6A-dependent manner. In human cultured MKs, TNS1 knockdown resulted in defective F-actin polymerization and assembly, impaired spreading on extracellular matrix proteins, and decreased proplatelet formation. Taken together, our research reveals the crucial functions of circRNAs in platelet production and has significant implications for the development of therapeutic strategies for thrombocytopenia and bleeding disorders.

Bruton's tyrosine kinase inhibitors (BTKi) and cell therapy have successfully been used to treat mantle cell lymphoma (MCL); however, therapy resistance inevitably emerges. Cancer cells can progressively develop stable resistance by traversing through a transient drug-tolerant persister (DTP) state. The mechanisms enabling DTP cells to reversibly adapt to therapies and evolve to acquire heterogeneity remain poorly understood, and characterizing DTP cells in MCL continues to pose a challenge for clinic translation. Here using pirtobrutinib, a recently FDA-approved non-covalent BTKi, we identified pirtobrutinib-tolerant persister cells exhibiting morphological variability by presenting a unique population of enlarged cells (Giant cells) with reversible fate transitions. During treatment, Giant cells enter a non-proliferative, dedifferentiated state, addicted to an activated cytosolic tricarboxylic acid (TCA) cycle coupled with the malate-aspartate shuttle to engage in biosynthesis. Upon drug removal, the TCA cycle shifts to oxidative catabolism, promoting Giant cells to differentiate into regular-sized cells. Throughout the transition, acetyl-CoA modulates cell fate by fine-tuning stemness. Our biphasic model demonstrates that the metabolic switch governs the phenotypic plasticity of DTP cells in MCL, resulting a dynamic presence of DTP cells across various developmental states in response to systemic therapies. Targeting Giant cells prior to their differentiation offers a promising strategy to overcoming therapy resistance in MCL.

Front-line pharmaceutical treatment for treatment of acute graft-versus-host disease (aGVHD) is not uniformly effective and has toxic side effects. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with potent in vitro and in vivo immunosuppressive functions. Clinical translation of in vitro generated MDSCs has been limited by the need for high MDSC:T cell ratios, multiple infusions to reduce inflammation and a relatively low peripheral blood-derived MDSC (PB-MDSCs) yield. To circumvent these obstacles, we developed a methodology to generate MDSCs using human induced pluripotent stem cell (iPSC)-derived CD34+ cells. Compared to PB-MDSCs, iPSC-MDSCs (iMDSCs) shared similar morphology, phenotype, and suppressive function. We found that the CD14+ iMDSC subset possessed the highest suppressor function. In previous studies, we reported that MDSCs transferred on day 0 into mice undergoing GVHD lost suppressor function due to inflammasome activation and immature myeloid cell maturation1. In striking contrast to human PB-MDSCs, we show here that iMDSCs retained 95% of suppressor function in vitro despite exposure to LPS+ATP, danger-associated molecular patterns inflammasome activating stimuli released early post-transplant during conditioning and GVHD-induced injury. When transferred in vivo with PB mononuclear cells, iMDSCs significantly increased recipient survival without loss of anti-leukemia effects. iMDSC RNAseq and gene knockdown studies revealed that the maintenance of the purine metabolizing enzyme, phosphoglycerate dehydrogenase, during LPS+ATP was linked to iMDSC inflammasome resistance. Taken together, these data provide a platform for translating in vitro generated, off-the-shelf iMDSCs into the clinic for suppressing a spectrum of adverse immune responses including GVHD.

Blood production is sustained by hematopoietic stem cells (HSCs), which are typically the only blood cells capable of long-term self-renewal. HSCs exhibit and depend on low levels of protein synthesis to self-renew. However, the mechanisms by which HSCs regulate protein synthesis to maintain their self-renewal capacity during proliferative stress and leukemogenesis remain unknown. Here we show CD99, a protein upregulated in leukemia stem cells (LSCs) in acute myeloid leukemia (AML), is required for the self-renewal of proliferating HSCs and LSCs. We found that loss of CD99 in HSCs and LSCs leads to increased protein synthesis and that their self-renewal capacity can be restored by translation inhibition. These data demonstrate a functional role for CD99 in constraining protein synthesis, which may promote the clonal expansion of HSCs and LSCs that leads to AML. Furthermore, these studies demonstrate that similar to HSCs, LSCs depend on maintenance of tightly regulated protein synthesis rates.

Cancer vaccines are emerging as promising therapies to not only prevent cancer but to treat cancer. Here, we developed a therapeutic vaccine for multiple myeloma (MM) using BCMA protein as a target. Given the remarkable efficacy of COVID 19 mRNA vaccines, we first packaged sequence- and base- optimized BCMA mRNA into lipid nanoparticles (LNPs) using next-generation ionizable lipid enhancing their accumulation in the spleen. A TLR3 agonist, polyinosinic:polycytidylic acid (Poly(I:C)), was also encapsulated in LNPs to further elicit BCMA-specific immune response. BCMA-mRNA LNPs were internalized by dendritic cells (DCs) in vitro, triggering proliferation and activation of BCMA-specific CD8+ cytolytic T cells (CTLs). Importantly, these CTLs lysed BCMA+ U266 MM cells and CD138+ patient MM cells, without affecting BCMA-knockout (KO) U266 or CD138- patient derived bone marrow cells. Vaccination of C57BL/6J mice with BCMA-mRNA LNPs activated splenic DCs and induced BCMA-specific CTLs, assessed by tetramer staining, which selectively killed murine 5TGM1 BCMA overexpressing (5TGM1-BCMA-OE) MM cells. Finally, vaccination of C57BL/KaLwRijHsd mice bearing BCMA-overexpressing 5TGM1 cells inhibited tumor growth associated with BCMA-specific CD8+ T cell responses. The combination treatment with Poly(I:C) further triggered the immune response induced by BCMA-mRNA LNPs in all instances. Our findings provide the framework for clinical evaluation of BCMA-mRNA LNP vaccines to improve patient outcome in MM.

Haemophilia A is an X-linked bleeding disorder caused by a blood clotting protein factor VIII deficiency. Patients with haemophilia develop recurrent bleeding episodes. When bleeding occurs in the joints, haemophilic arthropathy (HA) may develop, resulting in hemarthroses and joint deformation. A novel congenic mouse model of severe haemophilia A was generated using CRISPR/Cas9 targeting of exon-1 of the F8 gene (F8em1-/-) to explore changes in the bleeding and inflammation during HA. F8em1-/- mice have a high penetrance of spontaneous bleeding, with joint bleeds progressing to arthropathy. F8em1-/- mice were subjected to needle-induced damage to the knee to assess synchronised joint bleeding, and the development of HA and synovial inflammation was assessed. The synovium of injured joints of F8em1-/- mice had differential and temporal expression of inflammatory genes after injury. Pathway analysis identified upregulation of the IL-1 family cytokines, IL-1b and IL-33, and respective receptors IL-1RAP and T1/ST2 (ST2) in the synovium of mice after needle-induced HA. Soluble ST2 and IL-33 levels were elevated in the plasma of F8em1-/- mice in acute stages after needle injury to the joints. Dual ST2 deficient F8em1-/- mice were generated, with ST2 deficient haemophilic mice developing significantly reduced joint damage after needle injury relative to F8em1-/- mice. Using a therapeutic intervention, blocking ST2 following joint injury significantly ameliorated joint damage during HA in haemophilic mice. These studies in a new mouse model of HA identify a crucial role of ST2 in HA pathogenesis and highlight its potential as a novel therapeutic target.

RGI-2001, a liposomal glycolipid that binds CD1d receptor of antigen-presenting cells, can activate invariant natural killer T cells and stimulate cytokine-dependent proliferation of regulatory T-cells (Tregs). This open-label, single-arm, multicenter phase 2b trial evaluated the safety and efficacy of RGI-2001 in combination with standard graft-versus-host disease (GVHD) prophylaxis in participants receiving myeloablative allogeneic hematopoietic cell transplantation (HCT) for hematologic malignancies. RGI-2001 was infused at a dose of 100 ug/kg for six weekly doses starting on Day 0 of HCT. The primary endpoint was grades II-IV acute GVHD by Day 100 after HCT. Forty-nine participants received RGI-2001 in combination with tacrolimus and methotrexate. RGI-2001 was well tolerated, with no serious infusion reactions. Sixteen participants experienced grade ≥3 treatment-related adverse events, with the most common being decreased appetite, leukopenia, thrombocytopenia and stomatitis. The estimated probability of grades II-IV and III-IV acute GVHD were 24.9% and 4.1%, respectively. Compared to controls from the Center for International Blood and Marrow Research Transplant registry, participants receiving RGI-2001 experienced superior clinical outcomes, including Day-180 grades II-IV acute GVHD-free survival (70.8% vs 50.7%, adjusted hazard ratio 0.45, 95% CI 0.30-0.68). Increasing NKT and Treg populations were observed after HCT, consistent with the proposed action of RGI-2001. In conclusion, RGI-2001 was well tolerated and was associated with low rates of acute GVHD and encouraging survival after myeloablative HCT. These results support strategies that target NKT and Treg cell populations to augment immunological changes in allogeneic HCT recipients. This trial was registered at www.clinicaltrials.gov as NCT04014790.