Mutations in TP53 are mutually exclusive with other known drivers of myeloid transformation and define a distinct molecular subtype within de novo Acute Myeloid Leukemia (AML) that is associated with a complex karyotype, resistance to chemotherapy, and poor prognosis. Although TP53 defects are rare in de novo AML, biallelic mutations are a defining molecular feature of erythroleukemia. The genetic alterations that cooperate with defective TP53 to transform erythroid progenitors remain unknown. We found that loss of BAP1 (BRCA1 Associated Protein-1) co-occurs in one-third of patients with TP53-mutated AML, is associated with an erythroid-primed gene expression signature, and confers an additional adverse effect on overall survival. BAP1 is a tumor suppressor involved in the DNA damage response as well as epigenetic regulation through histone H2AK119 de-ubiquitination. While Bap1KO mice develop myelodysplasia with prominent dyserythropoiesis, combined deletion of Bap1 and Trp53 caused transplantable erythroleukemia, and occasionally mixed AML, mirroring the heterogeneity of human disease. Bulk and single-cell RNA-seq coupled to ChIP-seq in hematopoietic progenitors revealed that Bap1 loss triggers a proinflammatory response and cooperates with Trp53 deficiency to transform erythroid-primed multipotent progenitors. Mechanistically, genomic instability led to the development of erythroleukemia, while epigenetic deregulation caused myelomonocytic skewing suggesting a dichotomous and context dependent role for BAP1. We also demonstrate that BAP1 deficient erythroleukemia is dependent on BCL2L1 expression and is sensitive to BCL-xL inhibitors in vivo.

In inflammation, circulating neutrophils indirectly damage the skeleton by inducing formation of bone-resorbing osteoclasts. However, neutrophil progenitors in marrow have no known physiological function. A bone-protective role for the neutrophil lineage was recently suggested when a profound defect in bone structure was observed in mice with neutropenia due to Granulocyte Colony Stimulating Factor (G-CSF) deletion coupled with STAT3 hyperactivation in bone cells. Here, we tested the existence of this protective effect by manipulating neutrophil progenitors in bone marrow by Ly6G antibody (aLy6G) treatment. Two protocols revealed an inverse relationship between marrow neutrophil progenitors and osteoclasts. Two weeks of aLy6G treatment increased marrow immature neutrophils by 25% and halved osteoclast mRNA markers in cortical bone. In contrast, coupling six weeks of aLy6G with anti-rat IgG2a to maintain antigenicity reduced marrow pre-neutrophils by 50%. This doubled trabecular osteoclast surface, halved trabecular bone mass, and significantly reduced high density bone mass both in control mice, and in mice with bone-specific STAT3 hyperactivation. In culture, isolated pre-neutrophils dose-dependently inhibited osteoclastogenesis independent of direct contact. We conclude that neutrophil progenitors directly inhibit osteoclast formation by releasing soluble factors. This identifies a novel action of hematopoietic cells in marrow to protect bone structure.

Platelet shape and volume changes are early mechanical events contributing to platelet activation and thrombosis. Here, we identify single-nucleotide polymorphisms in Leucine-Rich Repeat Containing 8 (LRRC8) protein subunits that form the Volume-Regulated Anion Channel (VRAC) which are independently associated with altered mean platelet volume. LRRC8A is required for functional VRAC in megakaryocytes (MKs) and regulates platelet volume, adhesion, and agonist-stimulated activation, aggregation, ATP secretion and calcium mobilization. MK-specific LRRC8A conditional knockout mice have reduced laser-injury induced cremaster arteriolar thrombus formation and prolonged FeCl3 induced carotid arterial thrombosis without affecting bleeding times. Mechanistically, platelet LRRC8A mediates swell-induced cytosolic ATP release to amplify agonist-stimulated calcium-PI3K-AKT signaling. Small-molecule LRRC8 channel inhibitors recapitulate defects observed in LRRC8A-null platelets in vitro and in vivo. These studies identify the mechanoresponsive LRRC8 channel complex as an ATP release channel in platelets which positively regulates platelet function and thrombosis, providing a proof-of-concept for a novel anti-thrombotic drug target.

Disease-defining signatures in lymphomas, driven by intricate molecular mechanisms, have advanced molecular taxonomies, refined classification, and may guide clinical management; however, the role of these signatures in driving disease hallmarks including subtype-specific organotropism remains largely unexplored. Primary mediastinal large B-cell lymphoma (PMBCL) is an exemplary lymphoma characterized by disease manifestations in the thymic niche, unique genetic alterations and immune escape. Here, we identified IRF4-C99R mutations uniquely occurring in PMBCL through mutational meta-analysis of large-scale datasets. Our functional studies, integrating multi-omics approaches with genome editing in PMBCL cells, revealed that IRF4-C99R contributes to a differentiation block phenotype. Specifically, we showed that IRF4-C99R reduces its binding to the ISRE motif within PRDM1, which encodes a key transcriptional regulator of B-cell differentiation, resulting in decreased PRDM1 expression. Additionally, IRF4-C99R suppresses TNIK, a key IFNγ pathway regulator, by impairing ISRE motif binding, thereby reducing IFNγ signaling and increasing thymus and activation-regulated chemokine (TARC) expression, which drives TARC-mediated chemotaxis of T regulatory cells. We also revealed that IRF4-C99R upregulates Ephrin Type-B Receptor 1 (EPHB1) through non-canonical AICE motif binding, and showed that overexpression of EPHB1 in an immunocompetent syngeneic lymphoma model influenced organotropism to favor thymic localization, without affecting tumor burden in other organs. IRF4-C99R mutation-induced phenotypes were validated in primary PMBCL tissues using single-nuclei RNA sequencing, confirming that the molecular mechanisms observed in vitro align with the pathophysiology of PMBCL in patients. Together, these findings demonstrate how a single genetic mutation orchestrates the coordinated regulation of hallmark traits including thymus-specific tropism in PMBCL.

Anemia of inflammation (AI) is the second most common form of anemia and is prevalent in patients with chronic inflammatory states, such as infection, autoimmunity, and cancer. Interleukin 6 (IL6) is well-known to induce the iron-sequestering hormone hepcidin, which results in iron-restricted anemia. The contributions of other pro-inflammatory cytokines such as tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ) are less understood in the pathophysiology of AI. This study investigated the role of TNFα in a mouse model of AI by administering heat-killed Brucella Abortus (HKBA) to germline TNFα knockout (KO) mice. We hypothesized that TNFα possessed an important role in restoring steady-state erythropoiesis after inflammatory insult. TNFαKO injected with HKBA displayed a chronic anemia, with elevated pro-inflammatory IL12p40 and IFNγ cytokines that did not resolve. However, IFNγKO and TNFαKO/FNγKO double knock-out (DKO) mice showed reduced inflammation and anemia following HKBA administration. Since IFNγKO displayed normal serum TNFα and IL12p40 levels, we hypothesized that the persistent anemia was IFNγ-induced and TNFα was necessary for AI cessation. However, treatment with recombinant TNFα (rTNFα) accelerated death, while reducing IFNγ using an anti-IFNγ antibody (Ab) only briefly improved anemia. Only the combination of both the Ab and rTNFα together reversed the hyper-inflammatory phenotype, restored erythropoiesis, and prevented death of TNFαKO+HKBA mice. Our data provides compelling evidence for an anti-inflammatory role of TNFα that is necessary for the restoration of erythropoiesis and mitigation of pro-inflammatory IFNγ action in a mouse model of AI.

An acute inflammatory response to infection or sterile injury involves an adequate activation and recruitment of leukocytes. Activation of β2-integrins is required for neutrophil recruitment and is also mandatory for various neutrophil cell-intrinsic functions. GTPases are key regulators of the actin cytoskeleton and are required for β2-integrin activation. MyosinIXb (Myo9b), a Rho GTPase-activating protein, is essential for regulating Rho activity in neutrophils. Yet, the exact molecular mechanism how Myo9b regulates β2-integrin activity and neutrophil recruitment into inflamed tissue is unknown. We demonstrate that Myo9b deficiency causes RhoA overactivation, increases actin cytoskeleton rearrangement in neutrophils, decreases neutrophil recruitment into the kidney and improves kidney function in murine models of acute kidney injury. Loss of Myo9b also affects neutrophil effector functions and causes increased rolling velocity, decreased adhesion, impaired crawling, and strongly reduced transmigration of neutrophils in vivo. Mechanistically, Myo9b regulates RhoA activity which is required for chemokine- and selectin-induced talin-1 recruitment to β2-integrins. Thus, Myo9b is a crucial regulator of important signaling pathways in neutrophils and is required for an adequate immune response triggered by chemokines and selectins.

Rapid CD137 upregulation on alloreactive T-cells upon allogeneic stimulation suggests that their selective elimination could prevent acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (HCT). Here, we developed a novel aGVHD prophylactic regimen consisting of a single dose of an anti-CD137 antibody-drug conjugate (CD137-ADC) administered on the day of transplant without additional immunosuppression. The CD137-ADC depleted both human and non-human primate (NHP) activated T-cells and proved highly effective in preventing xenogeneic aGVHD in mice receiving human peripheral blood mononuclear cells (PBMC), as well as in NHP undergoing MHC-haploidentical HCT. Flow cytometry analysis of NHP T-cells indicated specific depletion of activated PD-1+ CD4 and CD8 T-cells, while sparing naïve and PD-1-OX40+ memory T-cell subsets during the first week after HCT. CD137-ADC-treated NHP recipients demonstrated robust hematopoietic and immune reconstitution. Hallmarks of T-cell recovery after CD137-ADC, which were associated with long-term aGVHD-free survival, included reconstitution of CD4 memory T-cells expressing TRAIL, terminally-differentiated CD8 T-cells expressing CX3CR1, and CD4 FoxP3+ Tregs - cell types not expected to be involved in aGVHD pathogenesis. CD137-ADC-treated recipients demonstrated a higher risk of reactivation of rhLCV (the rhesus macaque EBV analogue), which was associated with reconstitution of follicular helper T-cells, interferon signaling-associated memory, and gamma-delta T-cell subsets. This reactivation was controllable with rituximab administration. These results document effective depletion of alloreactive T-cells and prevention of aGVHD following a single dose of CD137-ADC, suggesting that clinical translation should be carefully explored.

Cellular therapies targeting B-cell maturation antigen have shown promise in controlled clinical trials, but their impact in broader, diverse patient populations remains underexplored. This study examines the real-world efficacy and safety in 343 triple-class exposed patients with relapsed and refractory multiple myeloma (RRMM) who received idecabtagene vicleucel (ide-cel, n=266) or ciltacabtagene autoleucel (cilta-cel, n=77) after more than three prior lines of therapy in Germany. Cilta-cel as compared to ide-cel, demonstrated superior outcomes, achieving a higher overall response rate (94% vs. 82%) and 10-month progression-free survival (PFS, 76% vs. 47%). Cilta-cel also led to higher complete response (CR, (61% vs. 39%) and improved response conversion, with more patients achieving CR after starting from less than CR pre-CAR T. For those attaining CR post-therapy, cilta-cel showed longer PFS, especially in patients who entered treatment with a partial response or worse. Cytokine release syndrome was observed in 85% of cilta-cel and 81% of ide-cel cases, predominantly low-grade. Immune effector cell-associated neurotoxicity syndrome was more common with cilta-cel (25% vs. 15%), although non-relapse mortality at 10 months was comparable between therapies (7% vs. 5%). Weighted multivariable analysis after propensity score matching confirmed significant advantage in terms of PFS for cilta-cel, with a hazard ratio of 0.48. Overall, outcomes in our registry analysis were comparable to the pivotal trials that led to approval of the respective agents. Cilta-cel demonstrated a greater capacity for response conversion and durable remission. These findings underscore the need for individualized CAR T therapy selection to optimize patient outcomes.

CD19-directed chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of relapsed/refractory B-cell non-Hodgkin lymphoma (B-NHL) and recently showed effects in autoimmune diseases such as systemic lupus erythematosus (SLE). Despite high levels of inflammation, toxicity appeared to differ in SLE and B-NHL. We therefore compared CAR T-cell kinetics and treatment-related side-effects to better define the distinct toxicity profiles. Contrary to similar CAR T-cell expansion, SLE patients revealed lower incidence and severity of cytokine-release syndrome, immune-effector cell-associated neurotoxicity syndrome, and immune-effector cell-associated hematotoxicity. While neutrophil nadir was lower in SLE patients after therapy, platelets remained close to normal and hematotoxicity was shorter in SLE than in B-NHL. Reduced hematotoxicity correlated with lower acute phase inflammation, better hematological reserve prior to CAR T-cell therapy, and distinct serum cytokine profiles. Interestingly, CAR T-cell persistence was consistently shorter and reconstitution of conventional T- and B-cells was faster in SLE. In both cohorts, B-cell reconstitution correlated with functional CD4+ T-cell recovery, indicating a general biological process of hematopoietic and immune system regeneration. In summary, similar lymphodepletion and CAR T-cell pharmacokinetics resulted in distinct toxicity, demonstrating a favorable side effect profile of CAR T-cell therapy in SLE including faster recovery of the adaptive immune system.