Highlights
Key points
– The combination of venetoclax (BCL‑2 inhibitor) and gilteritinib (FLT3 inhibitor) produced robust anti‑leukemia responses across diverse preclinical models of FLT3‑mutant, BCL11B‑activated lineage‑ambiguous leukemia.
– BH3 profiling implicated pre‑existing dependence on multiple anti‑apoptotic BCL‑2 family proteins as a determinant of venetoclax monotherapy resistance; combined FLT3 inhibition appears to re‑prime mitochondria for apoptosis.
– Longitudinal single‑cell RNA sequencing highlighted mitochondrial pathway activation and a pro‑lymphoid transcriptional program in rare treatment‑surviving cells, suggesting mechanisms for minimal residual disease and potential future resistance.
Background: clinical context and unmet need
Lineage‑ambiguous leukemias are rare hematologic malignancies characterized by co‑expression of markers and gene programs from multiple hematopoietic lineages. These diseases — which include cases with early T‑cell precursor (ETP) phenotypes and other mixed T‑lymphoid/myeloid presentations — are clinically heterogeneous and often respond poorly to standard lineage‑directed therapies. A subset of these leukemias is defined by aberrant activation of BCL11B (termed BCL11B‑a), which confers a stem/progenitor immunophenotype and a mixed lymphoid/myeloid gene expression profile.
Activating FLT3 mutations (e.g., internal tandem duplications or kinase domain mutations) frequently co‑occur with BCL11B‑a in these cases, providing a clear rationale for FLT3‑directed therapy. However, optimal targeted strategies for BCL11B‑a lineage‑ambiguous leukemia remain undefined. Venetoclax, a selective BCL‑2 inhibitor, has substantially changed care in several myeloid contexts (notably in combination with hypomethylating agents for older/unfit acute myeloid leukemia), and gilteritinib is an approved FLT3 inhibitor with demonstrated single‑agent activity in relapsed/refractory FLT3‑mutant AML. The biological interplay of anti‑apoptotic signaling and FLT3‑driven proliferation suggests that dual targeting might be synergistic, but this has not been systematically evaluated until recently.
Study design and methods
Montefiori and colleagues (Blood 2025) evaluated the therapeutic potential of venetoclax plus gilteritinib (VenGilt) in preclinical models of FLT3‑mutant, BCL11B‑activated lineage‑ambiguous leukemia. The study used a complementary set of experimental systems to capture disease biology and drug response dynamics:
– In vitro assays across multiple cell lines representative of the BCL11B‑a phenotype.
– Patient‑derived xenograft (PDX) and other in vivo murine models to assess anti‑leukemia efficacy and durability.
– BH3 profiling — a functional assay of mitochondrial apoptotic priming — to interrogate tumor intrinsic dependence on specific anti‑apoptotic BCL‑2 family proteins before and after therapy.
– Longitudinal single‑cell RNA sequencing (scRNA‑seq) to map transcriptional states of cancer cells during therapy and to characterize residual populations that survive dual treatment.
Primary endpoints in the preclinical context included measures of tumor burden, survival in xenografts, apoptosis induction, and transcriptional/functional correlates of sensitivity and resistance.
Key findings
Overall efficacy of the combination
– Across the preclinical models tested, VenGilt produced substantially greater anti‑leukemia activity than either venetoclax or gilteritinib alone. While single‑agent responses were variable between models, the combination uniformly produced deep cytoreduction and durable responses in vivo.
Mechanistic insights from BH3 profiling
– BH3 profiling revealed that baseline mitochondrial priming varied between models. Some leukemias were primarily BCL‑2 dependent and thus sensitive to venetoclax monotherapy; others showed dependence on alternative anti‑apoptotic proteins (notably MCL‑1 and/or BCL‑XL) and were resistant to single‑agent venetoclax despite similar FLT3 mutation status.
– The addition of gilteritinib appeared to shift the balance of pro‑ and anti‑apoptotic signaling toward a state permissive for BCL‑2–mediated apoptosis, effectively overcoming intrinsic resistance in venetoclax‑insensitive models. Mechanistically, FLT3 inhibition likely reduced pro‑survival signaling (e.g., downstream STAT5 or MAPK pathways) that maintains alternative BCL‑2 family dependencies, thereby re‑priming mitochondria for BCL‑2 inhibition to trigger cell death.
Single‑cell transcriptomics of residual disease
– Longitudinal scRNA‑seq identified small subpopulations of cells that persisted after VenGilt exposure. These rare survivors displayed upregulation of mitochondrial and oxidative phosphorylation pathways, suggesting metabolic adaptation as a survival mechanism.
– Surviving cells also exhibited an enriched pro‑lymphoid transcriptional signature despite treatment with agents selected for myeloid‑oriented targets, implicating lineage plasticity and lymphoid gene programs in residual disease biology.
Implications for resistance and relapse
– The convergence of mitochondrial pathway activation and a pro‑lymphoid state in residual cells points to two non‑mutually exclusive resistance axes: metabolic reprogramming (which can blunt apoptosis) and lineage switching/plasticity (which may allow cells to evade lineage‑biased treatments).
– These data argue that complete eradication in BCL11B‑a leukemias may require approaches that address both apoptotic thresholds (e.g., combinations targeting multiple BCL‑2 family proteins or adding agents that destabilize mitochondrial function) and lineage‑plastic subclones (via immunotherapy or lineage‑transparent cytotoxic regimens).
Safety considerations in translation
– The study was preclinical and did not provide a comprehensive safety profile for the combination. Potential overlapping toxicities — most importantly myelosuppression and infection risk — will need careful evaluation in early phase clinical studies. Known toxicities of venetoclax and gilteritinib (e.g., neutropenia, tumor lysis risk with venetoclax; transaminitis and QT effects with gilteritinib) should inform monitoring plans.
Expert commentary and mechanistic perspective
Biological plausibility
– The synergistic activity of venetoclax and gilteritinib in FLT3‑mutant, BCL11B‑a leukemia is consistent with established biology: oncogenic FLT3 signaling maintains proliferative and survival pathways that can sustain alternative anti‑apoptotic dependencies; inhibiting FLT3 removes survival cues and can lower the apoptotic threshold, thereby sensitizing cells to BCL‑2 inhibition.
– BH3 profiling, a functional assay of mitochondrial apoptotic readiness, provides a practical biomarker to explain heterogeneous responses and could be developed as a companion diagnostic to identify patients most likely to benefit from VenGilt.
Clinical precedent and analogies
– Venetoclax combinations have transformed management of older/unfit AML in combination with hypomethylating agents, demonstrating that combining BCL‑2 inhibition with agents that modulate survival signals can improve outcomes (DiNardo et al., N Engl J Med 2020).
– Gilteritinib monotherapy prolongs survival in relapsed/refractory FLT3‑mutant AML (Perl et al., N Engl J Med 2019). The current preclinical data suggest that combining these two targeted agents could be particularly effective in the biologically distinct subset of lineage‑ambiguous, FLT3‑mutant leukemias.
Limitations and unresolved questions
– The findings are preclinical and require confirmation in humans. Key uncertainties include tolerability of the combination (especially in heavily pretreated patients), optimal dosing and sequencing (concurrent vs. lead‑in), and durability of responses.
– The molecular basis of lineage plasticity and mitochondrial adaptations in residual cells requires further study; specifically, whether these changes are driven by selection of pre‑existing subclones or by therapy‑induced transcriptional reprogramming.
Translational roadmap: from bench to bedside
Priority elements for clinical translation
– Patient selection: trials should enroll patients with lineage‑ambiguous leukemia defined by BCL11B activation and FLT3 mutations. Where feasible, incorporate BH3 profiling and baseline scRNA‑seq as correlative studies to match biology to response.
– Early‑phase trial design: a phase 1/2 study evaluating safety, recommended phase 2 dose, pharmacodynamics, and preliminary efficacy of VenGilt is an appropriate next step. Cohorts might include newly diagnosed patients unfit for intensive chemotherapy and relapsed/refractory disease.
– Correlative endpoints: dynamic BH3 profiling, serial scRNA‑seq, monitoring for emergent FLT3 resistance mutations, and MRD assessment (flow cytometry and molecular) should be prioritized to understand mechanisms of response and resistance.
– Safety monitoring: proactive tumor lysis prophylaxis, intensive hematologic and infection surveillance, and ECG/liver function monitoring are essential given the known toxicity profiles.
Potential combination strategies and next steps
– For cases with residual MCL‑1 or BCL‑XL dependence, adding MCL‑1 inhibitors (under clinical development) or using agents that downregulate MCL‑1 might be rational. However, any triplet approach must be balanced against additive myelosuppression.
– Exploration of immune‑based consolidation — for example, antibody or cellular therapies that are lineage‑agnostic — could mitigate the risk from lineage‑plastic residual clones.
Conclusion
The preclinical study by Montefiori et al. provides compelling evidence that dual targeting of BCL‑2 and FLT3 with venetoclax plus gilteritinib achieves potent anti‑leukemia activity in FLT3‑mutant, BCL11B‑a lineage‑ambiguous leukemia models. Functional BH3 profiling and single‑cell transcriptomics offer mechanistic insights into variable monotherapy responses and the biology of residual disease. These data support prompt clinical evaluation, with careful trial design to define safety, optimal dosing, biomarkers of response, and strategies to prevent or overcome residual disease driven by mitochondrial adaptation or lineage plasticity.
Funding and clinicaltrials.gov
The study was published in Blood (Montefiori et al., 2025). Funding sources and disclosures are listed in the manuscript. As of publication, VenGilt has been recommended for clinical evaluation in this disease subset; there were no specific clinicaltrials.gov identifiers linked to a VenGilt trial in BCL11B‑a lineage‑ambiguous leukemia reported in the paper. Investigators and sponsors planning clinical translation should register subsequent trials on clinicaltrials.gov.
Selected references
– Montefiori LE, Iacobucci I, Gao Q, et al. Venetoclax plus gilteritinib is effective in preclinical models of FLT3‑mutant BCL11B‑a lineage‑ambiguous leukemia. Blood. 2025 Nov 6;146(19):2350‑2356. doi: 10.1182/blood.2025028985. PMID: 40811853; PMCID: PMC12377505.
– Perl AE, Martinelli G, Cortes JE, et al. Gilteritinib or chemotherapy for relapsed or refractory FLT3‑mutated AML. N Engl J Med. 2019;381(18):1728‑1740. doi:10.1056/NEJMoa1902688.
– DiNardo CD, Jonas BA, Pullarkat V, et al. Azacitidine and venetoclax in previously untreated acute myeloid leukemia. N Engl J Med. 2020;383(7):617‑629. doi:10.1056/NEJMoa2012971.
(higher‑resolution mechanistic and clinical references, BH3 profiling methodology, and additional prior work can be found in the Montefiori et al. manuscript and cited literature therein.)
