Hydrochloroquinine & Decitabine With Venetoclax in AML

Relapsed / refractory (R/R) AML remains a therapeutic dead end, with real-world series reporting a median overall survival of under six months, even with intensive salvage approaches. Although the hypomethylating-agent/BCL-2 inhibitor doublet of decitabine (now available as the oral cedazuridine-boosted formulation) plus venetoclax yields encouraging remission rates, most responders ultimately relapse, underscoring an urgent need for resistance-modifying strategies. Converging pre-clinical and translational data show that AML blasts upregulate cytoprotective autophagy to evade BCL-2-mediated apoptosis; pharmacologic blockade of autophagosome-lysosome fusion with chloroquine analogues reverses this escape and synergizes with both venetoclax and cytotoxic backbones. Hydroxychloroquine, the only orally available autophagy inhibitor with a long safety record, therefore represents a pragmatic, mechanism-based addition to an all-oral decitabine / venetoclax regimen. The proposed Phase I study is significant because it will be the first to co-target aberrant DNA methylation, intrinsic apoptosis, and adaptive autophagy in R/R AML, aiming to convert transient responses into durable remissions while preserving outpatient convenience. Success would not only fill a critical therapeutic gap for patients who otherwise face dismal prognoses but also provide clinical proof-of-concept that autophagy inhibition can extend the utility of venetoclax-based regimens across hematologic malignancies.

Venetoclax (VEN), a selective BCL-2 inhibitor, in combination with a hypomethylating agent (HMA) or low-dose cytarabine, has re-defined first-line therapy for older or unfit adults with acute myeloid leukemia (AML).In VIALE-A, azacitidine + VEN yielded an approximately 65% composite complete remission (CR + CRi) rate and a median overall survival (mOS) of 14.7 months versus 8 months with azacitidine alone. Despite these gains, the majority of responders ultimately progress; up to 42% relapse by two years in long-term follow-up of VIALE-A, and real-world datasets mirror these findings.[ Outcomes after VEN failure are dismal. A 2025 multicenter Spanish series reported an mOS of only 2.3 months following relapse or primary refractoriness to VEN-HMA, with a salvage treatment rate of 30% and an overall response rate (ORR) of 23% among those treated. Smaller US single-institution cohorts and ASH abstracts have described similar post-VEN mOS figures of 6 months and highlight the absence of a standard salvage approach.Thus, venetoclax-resistant relapsed/refractory (R/R) AML represents a high-mortality population with an urgent unmet need.

AML stem and progenitor cells are often in a state referred to as "primed" for mitochondrial, or intrinsic, apoptosis-meaning they are close to the threshold of undergoing programmed cell death. This apoptotic readiness is governed by the balance between pro-apoptotic and anti-apoptotic members of the BCL-2 protein family within the mitochondria. In many AML subtypes, particularly in leukemic stem-like cells, survival is heavily dependent on the anti-apoptotic protein BCL-2, which functions by binding to and sequestering key pro-apoptotic effectors such as BIM and BAX. BIM is a BH3-only activator protein, and BAX is a pore-forming effector that oligomerizes to disrupt the mitochondrial outer membrane, a critical step in the initiation of apoptosis.

VEN, a selective BCL-2 inhibitor, disrupts this protective interaction by displacing BIM from BCL-2. Once liberated, BIM is able to activate BAX and/or BAK, leading to mitochondrial outer membrane permeabilization (MOMP). This event triggers the release of cytochrome c into the cytosol, which binds APAF1 and forms the apoptosome, culminating in the activation of caspase-9 and downstream executioner caspases such as caspase-3 and caspase-7, the hallmark steps of intrinsic apoptosis. The result is rapid and irreversible cell death in BCL-2-dependent AML blasts.

Clinical and translational studies have shown that venetoclax sensitivity varies by AML genetic subtype. The highest response rates are observed in AML with NPM1 or IDH1/2 mutations, which often exhibit strong BCL-2 dependence and low expression of alternative anti-apoptotic proteins, such as MCL-1. Similarly, secondary AML with spliceosome mutations (e.g., SRSF2, U2AF1, or SF3B1) demonstrates heightened apoptotic priming and increased venetoclax responsiveness. Conversely, monocytic AML subtypes tend to upregulate MCL-1 and BCL2A1, reducing reliance on BCL-2 and conferring intrinsic resistance to venetoclax. Thus, understanding the apoptotic dependencies of AML subtypes has become critical in tailoring venetoclax-based therapies.

Acquired resistance to venetoclax in AML is driven by a multifactorial interplay of genetic, metabolic, and epigenetic mechanisms. One prominent mechanism involves clonal evolution and signal transduction pathway reprogramming. Longitudinal genomic analyses of patients at the time of relapse following initial response to venetoclax frequently reveal the emergence of mutations in key oncogenic drivers such as FLT3-ITD, NRAS, KRAS, and PTPN11. These mutations are not typically present at diagnosis but arise under the selective pressure of therapy. They lead to reactivation of downstream signaling pathways, most notably the MAPK cascade, which in turn upregulates anti-apoptotic proteins like MCL-1 and BCL-xL, thereby bypassing the BCL-2 dependence that venetoclax targets.

In addition to genetic adaptations, resistant AML blasts undergo profound metabolic rewiring, particularly within the mitochondria. These cells enhance their electron transport chain (ETC) capacity, replenish tricarboxylic acid (TCA) cycle intermediates, and activate antioxidant defense programs. This metabolic plasticity helps leukemic cells maintain mitochondrial integrity and survive even in the presence of BCL-2 inhibition, effectively diminishing venetoclax efficacy.

Finally, epigenetic reprogramming plays a significant role in acquired resistance. AML cells can dynamically remodel chromatin architecture - such as through changes in histone modifications like H3K27 acetylation - leading to a rebalanced expression of BCL-2 family proteins. These epigenetic changes shift the apoptotic threshold and may confer a resistance phenotype that can be reversible. Notably, agents targeting chromatin regulators, such as BETs (bromodomain and extraterminal domain) or LSD1 (lysine-specific demethylase 1) inhibitors, have demonstrated potential in restoring venetoclax sensitivity by reversing these epigenetic adaptations.

In response to the growing challenge of venetoclax (VEN) resistance in acute myeloid leukemia (AML), several innovative therapeutic strategies are being explored, both in preclinical models and early-phase clinical trials. One potentially promising approach involves directly targeting MCL-1, a key anti-apoptotic protein frequently upregulated in VEN-resistant AML. Selective MCL-1 inhibitors, such as S64315 (also known as MIK665), have demonstrated potent synergistic activity with venetoclax in preclinical studies, effectively eradicating resistant AML xenografts. Clinical evaluation is ongoing, with early-phase trials testing agents like AZD5991 (NCT03218683) and S64315 with or without venetoclax (NCT03672695). However, cardiac toxicity, particularly QT prolongation and ventricular dysfunction, remains a major safety consideration in this class.

Another promising avenue targets the Menin-KMT2A (formerly MLL) and NPM1 transcriptional axis, which contributes to leukemogenesis and BCL-2 family dysregulation.[ Small-molecule menin inhibitors such as revumenib and ziftomenib have shown the ability to restore myeloid differentiation and downregulate MCL-1 expression. Early clinical data in relapsed/refractory (R/R) AML patients previously exposed to venetoclax demonstrate complete remission or complete remission with partial hematologic recovery (CR/CRh) rates ranging from 20% to 40%, with encouraging durability of response when combined with venetoclax. However, these agents would only serve the approximately 35% of AML patients with NPM or KMT2A alterations.

To further enhance efficacy and prevent resistance escape, triplet regimens that concurrently inhibit compensatory signaling pathways are gaining traction. In patients with FLT3-mutated AML, the combination of a FLT3 inhibitor (e.g., gilteritinib or quizartinib) with a hypomethylating agent (HMA) and venetoclax has shown composite response rates exceeding 70%, including substantial activity in those with prior venetoclax exposure. These regimens are advancing to randomized phase III trials. Likewise, for RAS-mutated AML, preclinical studies suggest that dual targeting of MAPK signaling with trametinib and venetoclax induces synergistic cytotoxicity. This combination is currently being evaluated in phase I trials.

Modulation of the apoptotic pathway itself also represents a strategic target. SMAC mimetics (e.g., LCL161, ASTX660), which promote degradation of inhibitor of apoptosis proteins (IAPs), are under clinical investigation for their potential to re-sensitize venetoclax-resistant clones.Several of these agents are in phase Ib/II trials, independent of current frontline treatment protocols.

Finally, targeting the metabolic dependencies of AML cells is an emerging strategy aimed at reinforcing venetoclax cytotoxicity. Agents such as telaglenastat, a glutaminase inhibitor, and IACS-010759, a complex I inhibitor that blocks fatty acid oxidation, have shown preclinical efficacy in venetoclax-resistant AML by disrupting energy production and mitochondrial fitness and are in clinical testing.

Existing data show that post-VEN relapse/refractory AML carries an mOS of roughly 2-3 months, no approved therapies specifically address VEN-refractoriness. Despite the initial clinical efficacy of venetoclax and as discussed above, acquired resistance in acute myeloid leukemia (AML) remains a major barrier to durable remissions.[ Glytsou et al. have demonstrated that mitochondrial structural integrity and mitophagy are key elements of this resistance. Using a genome-wide CRISPR/Cas9 screens in human AML models identify mitochondrial organizing genes such as the mitochondrial chaperonin CLPB whose suppression sensitises AML cells to venetoclax.[44] They found that CLPB is upregulated in AML and further induced upon acquisition of Venetoclax resistance; mechanistically CLPB maintains mitochondrial cristae via interaction with the cristae-shaping protein OPA1. Loss of CLPB led to cristae remodeling, mitochondrial stress responses, and apoptosis, and synergised with ven and ven + aza. This work demonstrates mitochondrial architecture as a drug-gable vulnerability in venetoclax-resistant AML. Using multiple CRISPR/Cas9 loss-of-function screens and AML patient-derived xenograft models, they have also shown that overexpression of the mitochondrial fusion protein MFN2, enhanced mitochondria-ER contacts, and elevated mitophagy flux together allowed AML blasts to clear damaged mitochondria, thereby avoiding the mitochondrial outer membrane permeabilization (MOMP) triggered by BCL-2 inhibition. Inhibition (genetic or pharmacologic) of MFN2 or mitophagy regulators restored apoptotic sensitivity to BH3 mimetics, reducing leukemia burden in vivo. These data provide a mechanistic foundation for combining ven with mitophagy/mitochondrial-dynamics inhibitors as a rational salvage strategy.

Hydroxychloroquine is a lysosomotropic agent that accumulates in lysosomes, raises intralysosomal pH, and inhibits the fusion of autophagosomes (and mitophagosomes) with lysosomes.[In this way, HCQ effectively blocks the late stage of autophagy/mitophagy. In the AML context, this means that when mitochondrial damage is induced (for example by BCL-2 inhibition via venetoclax leading to mitochondrial outer membrane stress), the resistant AML cell cannot clear damaged mitochondria efficiently via mitophagy. The accumulation of damaged mitochondria leads to increased ROS, loss of mitochondrial membrane potential, disrupted cristae structure, and greater apoptotic priming. Thus, combining HCQ with venetoclax tilts the balance back toward mitochondrial apoptosis by disabling a resistance mechanism of enhanced mitophagy.

Therefore, we propose a study that will test hydroxychloroquine (HCQ) in combination with azacitidine in adults with venetoclax-resistant R/R AML. By selecting patients immediately at loss of VEN response, incorporating dynamic molecular profiling to confirm on-target pathway inhibition, and using composite CR with MRD negativity as the primary end-point, this trial seeks to define a new salvage backbone. Correlative assays will interrogate serial BCL-2 family protein dependencies, mitochondrial metabolism, and clonal evolution, directly addressing the biologic underpinnings outlined above. Given the bleak post-VEN prognosis and the mechanistic rationale for dual targeting, the study meets a pressing clinical need and is anticipated to inform future combinatorial strategies aimed at eradicating minimal residual disease and preventing early relapse in this high-risk population.

Inclusion criteria

• Confirmed acute myeloid leukemia per 2022 WHO (≥20 % blasts or genetically-defined AML) 2. Age ≥ 18 years on the date of consent Disease status 3. Phase I: Relapsed/refractory (R/R) or newly-diagnosed, overy high risk defined as p53 mutation or secondary AML after MPN or chemotherapy/radiation exposure 4. An ECOG performance status of 0-2 5. Acceptable Organ function within 14 days of cycle 1 day 1 6. AST/ALT ≤ 3× ULN; total bilirubin ≤ 1.5× ULN (unless Gilbert's) 7. Creatinine clearance > 60 mL/min (Cockcroft-Gault) 8. Subjects must have a baseline QTc interval of less than 450 milliseconds (<450 ms) White blood cells < 25 × 10⁹/L before first dose of hypomethylating agent - debulking with hydroxyurea/leukapheresis or up to 6 doses of cytarabine 100mg-200mg if needed is acceptable.

• HIV, HBV, and HCV patients are eligible if viral load is undetectable on stable therapy Wash-out from prior therapies should be ≥14 days from the last cytotoxic or targeted agent, and recovery to ≤Grade 1 non-hematologic toxicity 10. Reproductive precautions 11.Negative serum β-hCG for WOCBP; agreement to use highly effective contraception during treatment and ≥ 90 days after last dose Signed written informed consent and willingness to comply with study procedures

• Exclusion Criteria

• Leukemia subtype - Acute promyelocytic leukemia (APL, PML-RARA)
• CNS leukemia not cleared (<5 WBC/µL & no blasts), or symptomatic CNS involvement
• Recent or uncontrolled transplant-related complications Allogeneic HSCT ≤ 90 days before Day 1 Active grade ≥ 2 GVHD or systemic immunosuppression >10 mg/day prednisone-equivalent
• Concurrent malignancies requiring active therapy. Any adequately treated in-situ cancers or those not expected to interfere with endpoints are allowed.
• Active, uncontrolled infection (bacterial, viral, or fungal) despite appropriate antimicrobial therapy
• Cardiac risk, NYHA class III/IV heart failure, unstable angina, recent MI (< 6 months), clinically significant arrhythmia, Concomitant use of QT-prolonging medications that cannot be discontinued, or history of torsades de pointes
• Pregnant or breastfeeding.
• Patients with known G6PD deficiency.
• Investigational drug or major surgery within 28 days.
• Bleeding diathesis/coagulopathy that would preclude required marrow aspirates or lumbar puncture.
• Psychiatric or social condition that, in the investigator's judgment, would impair compliance with protocol-mandated visits/procedures.