Long-term efficacy and toxicity data are needed to inform optimal first-line treatment for individual patients with chronic lymphocytic leukemia (CLL). We report long-term outcomes of ibrutinib-venetoclax in older/comorbid patients at 67-month median follow-up of the phase III GLOW study (NCT03462719) and report grade 3/4 treatment-emergent adverse event (TEAE)-free progression-free survival (PFS). Patients were randomized 1:1 to ibrutinib-venetoclax (n = 106) or chlorambucil-obinutuzumab (n = 105). Sixty-six-month overall survival (OS) rates were 79.0% and 60.8% for ibrutinib-venetoclax and chlorambucil-obinutuzumab, respectively (hazard ratio [HR] 0.46). Ibrutinib-venetoclax reduced the risk of requiring second-line therapy by 77.4% and continued to improve PFS vs chlorambucil-obinutuzumab (66-month rates: 51.7% vs 18.1%, HR 0.27), regardless of IGHV/measurable residual disease (MRD) status. Ibrutinib-venetoclax-treated patients with undetectable MRD at end-of-treatment had better PFS outcomes than those with detectable MRD, especially in patients with unmutated IGHV. Grade 3/4 TEAE-free PFS was 21.4 months longer for ibrutinib-venetoclax vs chlorambucil-obinutuzumab (51.6 vs 30.2 months). Ibrutinib-venetoclax maintains superior OS, superior PFS, reduction in need for second-line treatment, and longer PFS duration without grade 3/4 TEAEs vs chlorambucil-obinutuzumab. Fixed-duration ibrutinib-venetoclax is an effective first-line treatment option for older/comorbid patients with previously untreated CLL, providing durable disease control and preserving future treatment options.

The receptor tyrosine kinase‑like orphan receptor (ROR1) is aberrantly expressed on chronic lymphocytic leukemia (CLL) cells and promotes survival signaling through NF‑κB, ERK1/2, and mTORC1. Zilovertamab is a humanized monoclonal antibody targeting ROR1. We conducted a multicenter phase 1b/2 study of zilovertamab plus ibrutinib (Z + I) in relapsed/refractory CLL. Phase 1b/2a (N = 34) established a recommended zilovertamab dose based on safety, pharmacokinetics, and ROR1 occupancy. In phase 2b, patients were randomized to zilovertamab plus ibrutinib (N = 18) or ibrutinib alone (I; N = 10); the primary endpoint was complete response (CR) per iwCLL, with progression‑free survival (PFS) and safety as secondary endpoints. Z + I was well tolerated, with no dose‑limiting toxicities and safety profile consistent with BTK inhibition. CR rates, overall response, and PFS were similar between randomized arms. In a post hoc subset of 10 patients with del(17p), Z + I was associated with prolonged disease control relative to historical BTK inhibitor experience; this hypothesis-generating observation requires prospective confirmation. Zilovertamab achieved ROR1 occupancy and attenuated NF‑κB, ERK1/2, and mTORC1 signaling in CLL cells, supporting further exploration of ROR1‑targeted strategies in biologically high‑risk CLL. The study was registered at ClinicalTrials.gov (NCT03088878).

Acute myeloid leukemia (AML) is characterized by complex molecular alterations including mutations in epigenetic regulators such as IDH1 and DNMT3A, which are associated with globally altered DNA methylation affecting gene transcription, treatment choices, and outcomes. Additionally, IDH1 mutations are linked to changes in DNA-loop formation leading to oncogene upregulation. Here, we assessed 3D-DNA conformational changes in IDH1- and DNMT3A-mutated (mut) AML on a genome-wide scale as a global driver of leukemic signaling to identify novel therapeutic vulnerabilities. Using a cellular model with either IDH1 p.R132H or DNMT3A p.R882H mutation and primary AML samples, we analyzed mutation-specific 3D-DNA architecture by chromatin-conformation-capture (HiC) and transcriptional alterations by RNA sequencing. We identified both shared and distinct changes in compartmentalization and underlying DNA-loop formation in both DNMT3A- and IDH1-mut AML, which were linked to differential gene expression, supporting that 3D-genome architecture broadly influences transcription. Thereby, loop-mediated upregulation of IGF1R was identified in IDH1-mut AML, corresponding to specific sensitivity to IGF1R inhibitor BMS-754807 as mono- and combination therapy with ivosidenib, while DNMT3A-mut AML exhibited sensitivity to p38/MAPK inhibitor ralimetinib. Our data present a comprehensive map of global 3D-DNA alterations associated with IDH1- and DNMT3A mutations as a basis for further assessment of novel therapeutic strategies for both AML entities.

Acquired non-genetic resistance mechanisms to existing therapies contribute to poor outcomes for acute myeloid leukemia (AML) patients, and the inability to target leukemic stem cells (LSCs) can lead to relapse. To overcome these challenges, we tested whether LSCs have dependencies on PI3- kinase (PI3K). We found that LSCs are susceptible to isoform-selective targeting of PI3K and are particularly dependent on the p110 alpha isoform of PI3K. We discovered that PI3K inactivation leads to dynamic changes in EZH2/PRC2 function in leukemic cells, and we uncovered downregulation of EZH2 protein levels as an adaptive resistance mechanism in response to PI3K inhibition. We found that PI3K inhibition in AML cells can lead to compensatory upregulation of EZH1, and that EZH1 knockdown can sensitize AML cells to PI3K inhibition. We leveraged this resistance mechanism by combining a PI3K inhibitor with an EZH1/2 dual inhibitor, which successfully overcomes the acquired resistance and leads to sustained targeting of AML cells ex vivo and in murine AML and PDX models in vivo. This study identifies a promising novel therapeutic regimen for targeting LSCs in AML.

Myeloproliferative neoplasms (MPNs) are clonal hematologic malignancies characterized by the overproduction of mature myeloid lineage cells. Although JAK2 inhibitors, such as ruxolitinib, can alleviate constitutional symptoms, they typically fail to eradicate malignant clones or reverse bone marrow fibrosis. Moreover, treatment failure and drug intolerance often limit their long-term use. Recent studies have identified PIM1 kinase as an important mediator of MPN pathogenesis. PIM1 expression is upregulated in MPN hematopoietic progenitors, which may contribute to aberrant proliferation and disease progression by regulating key downstream effectors, including mTORC1, BAD, MYC, HIF-1α, and TGF-β. Notably, PIM1 contributes to JAK2 inhibitor-persistent cell growth, and its inhibition restores ruxolitinib sensitivity in JAK2 mutant cells. Preclinical studies using genetic ablation and pharmacological inhibition of PIM1 have shown significant attenuation of the myelofibrosis phenotype in mouse models, providing a strong rationale for targeting this kinase. PIM kinase inhibitors are currently being evaluated in clinical trials for patients with myelofibrosis. This review summarizes the current understanding of PIM1 biology in the context of MPNs, details the molecular mechanisms underlying its pathogenic contributions, and evaluates the translational potential of PIM1-targeted therapies for MPNs.

Clonal monocytosis of undetermined significance (CMUS) and clonal cytopenia with monocytosis of undetermined significance (CCMUS) are recently defined precursor states, with unclear progression rates to myeloid neoplasms (MN), including chronic myelomonocytic leukemia (CMML). We evaluated a cohort of 958 patients with sustained monocytosis (≥ 0.5 ×10[9]/L, ≥10%) and stringently excluded MN. Among 958 patients, 74 met criteria for CMUS (n = 10) and CCMUS (n = 64), respectively. These patients were compared with a cohort with oligo monocytic-CMML (OM-CMML; n = 47). CMUS patients were younger (median age: 63 years), whereas CCMUS demonstrated lower BM blast %, in comparison to OM-CMML (median blast %: 1 vs. 2). OM-CMML was enriched for TET2 (60% vs. 32%, p = 0.012), ASXL1 (40% vs. 9%, p < 0.001), and SRSF2 (45% vs. 8%, p < 0.001) mutations, while CMUS/CCMUS had higher rates of DNMT3A (36% vs. 9%, p < 0.001) and PPM1D (12% vs. 0, p = 0.029) mutations. Mutation burden and VAF for most mutations (except SRSF2) were significantly higher in OM-CMML. DNMT3A mutations in CMUS/CCMUS often involved the methyltransferase (MT) domain, and MT domain mutations and co-mutations were associated with progression of CCMUS to CMML. With a median follow-up of 31 months, there were no progression events in CMUS patients, while 13% of CCMUS patients progressed to MN, mainly CMML (7/8). Median OS was inferior in OM-CMML (71 months) compared to CMUS/CCMUS (not reached), p < 0.001. Our findings demonstrate that CMUS and CCMUS represent biologically distinct clonal states with variable progression risk and highlight the importance of integrating morphologic and genomic data to refine classification.

Myeloproliferative neoplasms (MPN) are driven by the oncoproteins JAK2V617F, mutant calreticulin (CALR), and mutant thrombopoietin receptor (TPOR; MPL), all of which activate JAK/STAT signaling. While JAK2 signaling is engaged in all MPNs, TYK2 is dispensable for JAK2V617F-driven disease. Here, we hypothesized a distinct role for TYK2 in CALR-mutant driven MPN. We found constitutive TYK2 phosphorylation in CALRdel52/ins5- and MPLW515K- but not JAK2V617F-expressing cells. Structural modeling predicted similar TPOR-binding affinities for JAK2 and TYK2, while micropatterning experiments demonstrated that both JAK2WT and JAK2V617F displace TYK2 from the receptor. The TYK2 inhibitor deucravacitinib reduced viability and STAT3/5 phosphorylation in CALRdel52/ins5- and MPLW515K- but not JAK2V617F-mutant cells, with enhanced efficacy when combined with the JAK2-selective inhibitor fedratinib. Cellular response correlated with JAK2 protein abundance, as CALRins5 JAK2[high] clones outcompeted JAK2[low] clones upon TYK2 inhibition. In primary samples, deucravacitinib significantly suppressed colony growth in ET and PMF but not PV, and selectively reduced CALR- but not JAK2V617F-mutant allele burden. Similarly, CALR-mutant patient-specific iPSC-derived CD34[+] progenitors showed increased TYK2 phosphorylation and were more sensitive to TYK2 inhibition than their JAK2V617F counterparts. These findings identify TYK2 as a selective vulnerability in CALR-mutant MPN and support combined TYK2/JAK2 inhibition strategies to overcome JAK2-dependent resistance.

Although therapies targeting BTK of BCR-signal are changing the landscape of CLL management, these agents are not curative and resistance can develop. Thus, we searched for other druggable target(s) regulating leukemic B-cell survival for developing alternative therapies. We detected MERTK, a member of the TAM (Tyro3, AXL, MERTK) family receptor tyrosine kinases (RTKs), is aberrantly expressed and constitutively active in CLL cells from ~65% CLL patients (n = 100). Ligation of Gas6, a common ligand for TAM-RTKs, preferentially activated MERTK resulting in activation of NF-κB/AKT/Erk1/2 and BTK in CLL cells. Partial depletion of MERTK in CLL cells reduced P-Erk1/2, P-NF-κB, P-AKT, and P-BTK levels, suggesting that MERTK may crosstalk with BCR-signal. Indeed, MERTK activated BTK by forming a signalosome with CD79a/LYN in CLL cells. Interestingly, stimulation of BCR-signals in CLL cells activated MERTK via CD79a, suggesting that MERTK/BCR-signals are functionally connected. Importantly, targeting MERTK using a high-affinity, pharmacologic inhibitor UNC-2025 induced apoptosis in CLL cells and reduced leukemic burden in vivo. More significantly, ibrutinib-resistant CLL cells expressed high-levels of MERTK and that, UNC-2025 synergistically induced apoptosis when combined with venetoclax. Thus, high MERTK-levels in CLL cells may be associated with ibrutinib-resistance underscoring the potential use of UNC-2025/venetoclax combination in treating relapsed/refractory patients.

We recently reported that the functional intracellular complement network, known as the complosome, is expressed in hematopoietic stem/progenitor cells (HSPCs). In our recent work, murine lineage-negative (Lin[-]) bone marrow (BM) mononuclear cells (BMMNC) from C3-KO and C5[-/-] mice showed defects in oxygen consumption rate (OCR) and elevated lactate production, along with increased lactate dehydrogenase (LDH) release, indicating dependence on anaerobic glycolysis. Simultaneously, mitochondria from C3 and C5 complosome mutant Lin-BMMNC accumulated fewer MitoTracker probes, suggesting mitochondrial defects. To better understand these changes, we focused on the expression of (i) key enzymes involved in aerobic and anaerobic glycolysis, (ii) genes involved in mitochondrial quality control (MCQ), (iii) the electron transfer chain (ETC) activity of all five ETC complexes, and (iv) TEM analysis of mitochondrial morphology in purified murine Lin[-] BMMNC. Our data confirm that the complosome is important for maintaining proper mitochondrial development and function, as C3 and C5 mutants exhibit severe defects in complex I and III activity and display an increase in the number of immature and damaged mitochondria. To our knowledge, this is the first study conducted on cells enriched in HSPCs to directly demonstrate functional defects in the ETC in C3- and C5 complosome-deficient cells. These data are crucial for all studies that use C3- and C5-deficient mice to investigate immune responses, tissue and organ regeneration, aging, or behavioral processes, and provide solid evidence for a pivotal novel role for innate immunity in regulating hematopoiesis.

Assessment of fibrosis is central to the evaluation of diagnostic bone marrow trephine (BMT) biopsies. However, manual fibrosis grading is subjective and only semi-quantitative. We evaluated the clinical utility of a previously developed AI-based quantitative fibrosis assessment tool, Continuous Indexing of Fibrosis (CIF), using ~1000 consecutive BMT biopsies without pre-selection. An international panel of 14 haematopathologists performed manual reads using whole-slide images (WSI) of reticulin-stained slides and two types of CIF-assisted reads (Sequential-assisted and Concurrent-assisted) across three study rounds. The AI-derived CIF scores correlated strongly with the manual consensus MF grade (Spearman ρ = 0.770) and demonstrated good discriminative performance across adjacent MF grade boundaries. The CIF-assisted protocols significantly improved intra-observer and inter-observer agreement compared to manual assessment, without increasing read times. Sequential-assisted reads yielded the largest gain in inter-observer agreement (12.6 percentage points; 95% CI 10.5-14.8) and improved agreement with the consensus reference (3.0 percentage points; 95% CI 0.4-5.7). Both assisted protocols reduced discordance across the clinically significant MF-1 / MF-2 boundary. These findings demonstrate that AI-assisted quantitative bone marrow fibrosis assessment using CIF is reproducible, efficient and well suited for future clinical deployment testing. This supports a future role in standardising routine diagnostic haematopathology and enhancing the sensitivity of fibrosis-based clinical trial endpoints.

CD38 is a transmembrane glycoprotein highly expressed in acute myeloid leukemia (AML) and T-cell acute lymphoblastic leukemia (T-ALL). XmAb18968 is a novel CD38-CD3 bi-specific T-cell engager with Fc domain modified to reduce non-selective activation of effector cells. In this phase 1 multicenter clinical trial, we evaluated the outcomes of XmAb18968 in adults with relapsed/refractory (RR) AML and T-ALL. Twenty-two patients with AML (n = 13) and T-ALL (n = 9) with a median age of 63 years (range 31-77) were enrolled. Prior lines of therapy (median 3, range 1-8) included venetoclax (77.3%), allogeneic HCT (22.7%), CD7 CAR-T cell therapy and daratumumab (11.1%). Grade ≥3 adverse events included anemia (14%), neutropenia (18%), and thrombocytopenia (14%). No grade ≥3 cytokine release syndrome or neurotoxicity was seen. Overall, 17 patients (AML = 11, ALL = 6) completed at least one cycle of therapy. Among 11 patients with RR-AML, one achieved partial remission (PR) and two achieved MRD negative complete remission (CR). In 6 patients with RR-T-ALL, 4 achieved meaningful improvement in disease burden with 1 clearance of MRD. Median OS was 8.5 months in AML and 8.7 months in T-ALL. XmAb18968 is safe and tolerable with encouraging preliminary efficacy, supporting further investigation of CD38 targeting therapies in this setting (NCT05038644).