Recent clinical trials in both transplant eligible and ineligible newly diagnosed multiple myeloma utilising three and four drug combinations have demonstrated unprecedented levels of response. However, two recently published studies redefining high-risk in newly diagnosed multiple myeloma in the context of these newer and more effective treatments demonstrate that a significant minority of patients likely derive little benefit from these newer approaches. These new prognostic systems thus provide an evidence-based framework for the development of much needed risk-stratified clinical trials.

Chronic active Epstein-Barr virus (EBV) infection (CAEBV) is an orphan disease characterized by the proliferation and infiltration of EBV-infected T/natural killer (NK) cells into multiple organs. Although CAEBV is a heterogeneous disease with diverse clinical courses, its pathogenesis remains poorly understood. In this study, we explored the molecular mechanisms underlying CAEBV by performing a comprehensive multi-omics analysis, including genome, transcriptome, epigenome, and single-cell transcriptome and surface proteome analyses, of 65 CAEBV patients. Methylation analysis identified two distinct subtypes of NK cell-type CAEBV based on the CpG island methylator phenotype (CIMP). In CIMP-positive CAEBV, regions associated with enhancer of zeste homolog 2 binding sites and histone H3 lysine 27 trimethylation exhibited increased DNA hypermethylation, resulting in downregulation of tumor suppressor and anti-herpes virus genes. CIMP-positive CAEBV had a particularly poor prognosis and displayed a "neoplastic" phenotype with a DNA methylation pattern similar to that of extranodal NK/T-cell lymphoma, a higher tumor mutation burden, and frequent copy number alterations. In addition, both in vitro and in vivo functional assays demonstrated that 5-Azacytidine, a hypomethylating agent, was a potentially effective agent for high-risk CIMP-positive CAEBV. Finally, we established a method to effectively detect EBV-infected cells in single-cell analysis, suggesting that EBV-infected NK cells have tissue-resident properties and that innate and adaptive immunity to EBV is compromised in patients with CAEBV. The present findings provide insight into the complex molecular features of CAEBV and suggest potential molecular therapies.

TP53 mutated myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are among the most aggressive and chemotherapy refractory myeloid neoplasms with a median overall survival of less than 6 months. An enormous unmet need exists to develop novel therapeutic strategies and understand resistance mechanisms to suboptimal existing therapies for this disease. In two parallel phase 2 clinical trials that combined eprenetapopt with azacitidine in TP53 mutated MDS/AML, we observed complete remission rates of 40-50% and molecular remission rates of 38%. However, unless allogeneic stem cell transplantation was performed, relapse inevitably occurred. To understand the mechanisms of secondary resistance responsible for this, we genotyped sequential clinical trial samples, conducted a genome-wide CRISPR screen in TP53 mutated leukemia cells, and identified XPO1 as a therapeutically tractable mediator of resistance. We demonstrate that XPO1 is overexpressed in patient samples after eprenetapopt and azacitidine treatment, elucidate the mechanism by which this occurs, and determine that it is necessary and sufficient for resistance to combination therapy. Finally, we validate in a variety of model systems including a novel patient derived xenograft model of TP53 mutant MDS, that eprenetapopt in combination with XPO1 inhibitors can overcome this resistance, providing preclinical rationale that this novel combination strategy is a viable therapeutic approach in TP53 mutant MDS/AML patients.

With up to 10 years of follow-up, we report results from the final analysis of RESONATE-2 (NCT01722487/NCT01724346), a phase 3 study of first-line ibrutinib versus chlorambucil for the treatment of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Patients aged ≥65 years with previously untreated CLL/SLL without del(17p) were randomly assigned to receive either single-agent ibrutinib (420 mg/day; n = 136) or chlorambucil (0.5-0.8 mg/kg ≤12 cycles; n = 133) until disease progression/unacceptable toxicity. With a median follow-up of 9.6 in the ibrutinib arm, median PFS was 8.9 years (95% CI, 7.0-NE) versus 1.3 years (95% CI, 0.9-1.6) for the chlorambucil arm. Among patients with unmutated IGHV, del(11q), mutated TP53, or complex karyotype median PFS was 8.4 years (95% CI, 6.8-NE) with ibrutinib and 0.7 years (95% CI, 0.4-1.2) with chlorambucil. Median overall survival (OS) with ibrutinib was not reached. Most common adverse events (AEs) of any grade included diarrhea (52%), fatigue (41%), cough (39%), nausea (32%), arthralgia (31%), peripheral edema (31%), and hypertension (30%). During the entire study period, 34/136 patients (25%) had an ibrutinib dose reduction due to AEs; these AEs improved in 30/34 patients (88%). At study completion, 27% of patients remained on first-line ibrutinib treatment. With the longest follow-up to date from a phase 3 study of any targeted CLL/SLL therapy, this landmark RESONATE-2 study defines median PFS and demonstrates continued OS benefit of first-line ibrutinib treatment for patients with CLL/SLL, including those with high-risk genomic features. Sustained efficacy and tolerability of ibrutinib reemphasize the favorable benefit-risk profile.

Attempting to induce a complete remission (CR) prior to allogeneic hematopoietic cell transplantation (alloHCT) is current practice in patients with AML. A benefit of remission induction prior to alloHCT, however, has never been proven in a prospective trial. Potent conditioning regimens exist which allow for successful alloHCT in patients with active AML. Therefore, the ASAP trial was conducted to test remission induction by salvage chemotherapy prior to alloHCT against immediate transplantation after intensified conditioning. In total 281 patients with AML with poor response after first induction or untreated first relapse were randomized 1:1 to remission induction (RIST) with high-dose cytarabine plus mitoxantrone versus immediate alloHCT with sequential conditioning after non-intensive disease control measures (DisC) preferentially watchful waiting only. Overall survival (OS) at 5 years from randomization analyzed according to ITT was 46.1% for DisC versus 47.5% for RIST (p=0.82). In multivariable Cox regression analysis, genetic AML risk according to ELN (p<0.0001), age (p=0.001) and comorbidities (p=0.046) predicted survival, but not treatment arm (HR 1.08 for DisC versus RIST, p=0.67). In conclusion, long-term follow-up of the ASAP trial showed no survival advantage for standard salvage chemotherapy prior to alloHCT as opposed to immediate alloHCT. The trial results question the general concept of remission induction with intensive standard salvage therapy prior to alloHCT for all patients, since immediate alloHCT may reduce time in hospital and health care expenses. Well tolerable novel bridging therapies and post-transplant maintenance with targeted drugs are urgently warranted, especially for adverse-risk AML, to improve outcome after alloHCT. NCT02461537.

JAK2V617F is one of the most common mutations in clonal hematopoiesis of indeterminate potential (CHIP) and a major driver of myeloproliferative neoplasms (MPN). To determine the impact of a low-frequency of JAK2V617F clone on both the hematopoietic system and the bone marrow (BM) stroma, we developed a traceable murine MPN model, where the whole BM transplantation (BMT) was performed using CD45.2 5.0x106 JAK2V617F donor cells into unconditioned CD45.1 recipient mice. BMT recipients developed a polycythemia vera (PV)-like phenotype (elevated hematocrit and leukocytosis) with a 2.7% average donor cell chimerism in peripheral blood. Eight months post-BMT, RNA-seq analysis of the BM sorted according to CD45.1/CD45.2 expression, showed significant upregulation of early erythroblast- and myeloid cell-specific transcripts, as well as downregulation of lymphoid transcripts in donor-derived cells compared to controls. Surprisingly, recipient-derived cells also showed upregulation of myeloid- and erythroblast-related transcripts, indicating a skewing of the non-JAK2V617F carrying recipient hematopoietic system towards an MPN-like phenotype. In addition, RNA-seq analysis of the BM stroma from JAK2V617F BMT recipients indicated significant loss of osteo-mesenchymal transcripts. Consistently, micro-CT imaging indicated loss of trabecular bone. In sum, our results indicate that the low frequency MPN-driving cells in unconditioned recipients not only impact hematopoiesis-supporting stroma but profoundly influences unmutated cells, uniquely altering their transcriptomic and phenotypic profiles. These observations are challenging our current understanding of etiology and therapeutic approaches to MPNs and other CHIP-associated diseases.

TP53 mutations are found in 10-15% of myeloid neoplasms and are one of its most important prognostic factors. Emerging data show that TP53 mutational allele status is a key determinant of clinical outcomes, with multi-hit TP53 mutant myeloid neoplasms having a very poor prognosis. Significant differences exist among the methods used in clinical and research settings to assess TP53 mutational status, leading to variability in reported patient characteristics, response to therapy, and survival. Indeed, differences in the criteria used to define TP53 mutational states among professional societies and in landmark research studies have led to confusion, suboptimal clinical testing, and variability in therapy recommendations. We review the methods used to assess for TP53 mutational allele status and provide recommendations, based on clinically available testing, for the accurate evaluation of TP53 gene mutations in myeloid neoplasms. Hotspot mutations represent ~35% of all TP53 missense mutations in myeloid neoplasms. There is evidence that these hotspot mutations may have dominant-negative or gain-of function properties. Here, we review this evidence and discuss the potential impact of TP53 mutation identity on patient outcomes and clinical management.

The treatment landscape for chronic lymphocytic leukemia (CLL) has been revolutionized by the advent of novel agents, particularly covalent BTK inhibitors (cBTKis) and BCL-2 inhibitors (BCL-2is). This has resulted in significant improvement in outcome of patients with CLL many of whom experience a life expectancy comparable to general population. However, patients who are double-refractory, having progressed following exposure to both classes face limited options and poor outcomes. This manuscript presents a practical approach to managing double-exposed or double-refractory CLL, integrating clinical case discussions, trial data, and expert insights. For patients with intolerance to cBTKis, second-generation agents may remain effective. Similarly, re-treatment with venetoclax can be considered after prior fixed-duration use. In double-refractory disease, the non-covalent BTK inhibitor (e.g., pirtobrutinib) and CD19-directed CAR-T therapy (lisocabtagene maraleucel) are available standard-of-care options. Pirtobrutinib provides rapid disease control but often with limited durability, emphasizing the importance of early planning for consolidation with CAR-T or allogeneic stem cell transplant. Persistent disease after CAR-T warrants close follow-up and timely referral for transplant evaluation in eligible patients. While PI3K inhibitors are also available, their role is limited due to toxicity and modest efficacy. Investigational agents-including BTK degraders, bispecific antibodies, and novel cellular therapies-offer promise for the future. A nuanced, individualized treatment strategy that incorporates current therapies and emerging options is essential to optimize outcomes in double-refractory CLL.

VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is an autoinflammatory disorder caused by acquired somatic UBA1 mutations in hematopoietic stem cells, affecting peripheral myeloid and natural killer (NK) cells. Given the high rate of severe infections observed in VEXAS patients, we hypothesized that NK cell dysfunction contributes to this increased susceptibility. We conducted a comprehensive immune characterization of peripheral NK cells in patients with VEXAS (n=40), patients with autoinflammatory diseases without UBA1 mutations (n=22), and elderly gender-matched healthy controls (HCs) (n=16). Multiparameter phenotyping was performed using CyTOF, scRNAseq, whole blood stimulation assays and in vitro NK cell cytotoxic assay. Peripheral NK cells in VEXAS were quantitatively and qualitatively impaired. Mass cytometry revealed reduced frequencies of mature cytotoxic CD56dim NK cells and an expansion of the CD56high CD16dim subset. NK cells exhibited features of exhaustion, including increased PD-1 expression, and reduced levels of cytotoxic markers such as NKp46 and CD8α. scRNAseq analysis showed decreased signatures of cytotoxicity and IL-2 and IFN-γ production, alongside increased inflammatory signatures. Whole blood stimulation assays confirmed impaired IL-2, IFN-γ, and granzyme B production following TLR3, TLR4, and TLR7/TLR8 agonist stimulation. Extended NK phenotyping by flow cytometry confirmed reduced activating receptors' expression and impaired IFN-γ production in VEXAS. Moreover, in vitro UBA1 inhibitors impaired NK cell cytotoxic capacity and promote cell death. Finally, reduced NK cell frequencies were independently associated with an increased risk of severe infections. These findings suggest that NK cell dysfunction in VEXAS syndrome contributes to increased susceptibility to severe infections.

Throughout thrombopoiesis megakaryocytes (MKs) form proplatelets within the bone marrow (BM) and release platelets into BM sinusoids. Casein Kinase 1α (CK1α) is a major player and, thus an important therapeutic target in several hematological malignancies. This study aims to define the role of CK1α for the essential steps of thrombopoiesis and to dissect potential mechanisms of thrombocytopenia. MK-specific CK1α-deficiency resulted in a macrothrombocytopenia. Ck1αPf4Δ/Pf4Δ mice displayed a substantial BM hyperplasia with pivotal changes in MK nuclear lobulation and reduced contact to BM sinusoids. Ck1αPf4Δ/Pf4Δ MKs displayed a defective cytoskeleton organization reflected by a decreased amount of polymerized F-actin and disturbed microtubule dynamics due to p21/p53 accumulation and impaired ROCK/LIMK/cofilin signaling. Further, pronounced defects in DMS polarization and proplatelet formation of Ck1αPf4Δ/Pf4Δ MKs unraveled CK1α as a prerequisite for thrombopoiesis. Our findings could be translated into a human approach, since a CRISPR/Cas9-mediated genetic deletion of CSNK1A1 in MKs derived from human CD34+ progenitor cells resulted in a substantial defect in human MK maturation and platelet production. The present observations elucidated CK1α as an important signaling molecule in MK cytoskeletal dynamics and polarization, proplatelet formation and polyploidization, thus highlighting the crucial role of CK1α in platelet biogenesis.

Genetic alterations alone cannot account for the diverse phenotypes of cancer cells. Even cancers with the same driver mutation show significant transcriptional heterogeneity and varied responses to therapy. However, the mechanisms underpinning this heterogeneity remain under-explored. Here, we find that novel enhancer usage is a common feature in acute lymphoblastic leukemia (ALL). In particular, KMT2A::AFF1 ALL, an aggressive leukemia with a poor prognosis and a low mutational burden, exhibits substantial transcriptional heterogeneity between individuals. Using single cell multiome analysis and extensive chromatin profiling, we reveal that much transcriptional heterogeneity in KMT2A::AFF1 ALL is driven by novel enhancer usage. By generating high resolution Micro Capture-C data in primary patient samples, we identify patient-specific enhancer activity at key oncogenes such as MEIS1 and RUNX2, driving high levels of expression of both oncogenes in a patient-specific manner. Overall, our data show that enhancer heterogeneity is highly prevalent in KMT2A::AFF1 ALL and may be a mechanism that drives transcriptional heterogeneity in cancer more generally.

The purpose of this study was to explore and determine the optimal landmark for defining complete remission after intensive induction therapy that best correlates with long-term survival outcome among patients with newly diagnosed acute myeloid leukemia (AML).

This phase II trial assessed CD7 CAR T-cells derived from previous transplant or newly HLA-matched donors for relapsed/refractory T-cell acute lymphoblastic leukemia/lymphoma. It was designed to enroll 70 patients but terminated early, with 55 ultimately treated, due to discontinuation of departmental operations. Within three months, 89% (n=49) achieved best overall response of partial remission or better. 19 received stem-cell transplantation (SCT) at a median 1.3 months (range 1.0-10.6). After a 26.3-month median follow-up, median event-free survival was 5.0-months (95% CI: 4.1-8.4) with median 8.5-month overall survival (95% CI: 6.1-15.6). No deaths occurred in first 30 days; adverse events (AEs) included cytokine release syndrome in 87% (n=48) at grades 1-2 and 11% (n=6) at grade 3, neurotoxicity in 9% (n=5) at grade 1. Graft-versus-host disease (GVHD) was in 38% (n=21) at grades 1-2 and 2% (n=1) at grade 3. Grade 1-2 infections occurred in 29% (n=16). Cytopenias in 4% (n=2) at grades 1-2 and 96% (n=53) at grades 3-4. After 30 days, grade 3-5 AEs included cytopenias (grade 3 in 24%, grade 4 in 67%), infections (grade 3 in 9%, grade 4 in 5%, grade 5 in 9%), GVHD (grade 3 in 4%, grade 5 in 4%), thrombotic microangiopathy (grade 5 in 4%), and hepatic failure (grade 5 in 2%). 20% (n=11) encountered non-relapse mortality after 30 days, accounting for 35% of responders without consolidatory SCT. While effective at inducing remission, death in remission beyond 30 days is a concern. NCT04689659.