Supplementary Table S5 shows sgRNA, primers, shRNA sequences used in this study.

Supplementary Table S2 shows the correlation score of MFN2 expression with the ex vivo drug response (IC50, BeatAML data).

Supplemental Figure S1 shows the integration of CRISPR/Cas9 loss-of-function screens to identify dependencies and liabilities in BH3-mimetics treatments. Supplemental Figure S2 demonstrates that increased mitochondria-ER interactions contribute to BH3-mimetics resistance. Supplemental Figure S3 shows that mitophagy affects the responsiveness of AML cells to BH3-mimetics. Supplemental Figure S4 reveals that enhanced autophagic clearance of mitochondria as a mechanism of resistance to BH3-mimetics in AML. Supplemental Figure S5 exhibits the synergism between BH3-mimetics and macroautophagy inhibition in human AML. Supplemental Figure S6 shows that deletion of MFN2 or MARCH5 sensitizes AML cells to BH3-mimetics. Supplemental Figure S7 displays that targeting of MFN2 or MARCH5 impairs the process of mitophagy.

Supplementary Table S4 shows autophagy related genes and their ranking in NMF analysis of scRNAseq data.

Supplementary Table S3 shows the information of the AML patient samples used in this study.

Supplementary Table 1 shows the results of our CRISPRi screens.

BACKGROUND:Hypomethylating agents are approved in higher-riskmyelodysplastic syndromes. The combination of a hypomethylating agent with venetoclax is standard of care in acute myeloid leukaemia. We investigated the safety and activity of the first totally oral combination of decitabine plus cedazuridine and venetoclax in patients with higher-risk-myelodysplastic syndromes and chronic myelomonocytic leukaemia. METHODS:We did a single-centre, dose-escalation and dose-expansion, phase 1/2, clinical trial. Patients with treatment-naive higher-risk-myelodysplastic syndromes or chronic myelomonocytic leukaemia (risk level categorised as intermediate-2 or higher by the International Prognostic Scoring System) with excess blasts (>5%). Treatment consisted of oral decitabine 35 mg plus cedazuridine 100 mg on days 1-5 and venetoclax (variable doses of 100-400 mg, day 1 to 14, 28-day cycle). The primary outcomes were safety for the phase 1 part and the overall response for the phase 2 part of the study. The trial is ongoing and this analysis was not prespecified. This study is registered with ClinicalTrials.gov, NCT04655755, and is currently enrolling participants. FINDINGS:Between Jan 21, 2021, and Jan 20, 2023, we enrolled 39 patients (nine in phase 1 and 30 in phase 2). The median age was 71 years (range 27-94), 28 (72%) patients were male, and 11 (28%) were female. The maximum tolerated dose was not reached, and the recommended phase 2 dose was established as oral decitabine 35 mg plus cedazuridine 100 mg for 5 days and venetoclax (400 mg) for 14 days. The most common grade 3-4 adverse events were thrombocytopenia (33 [85%] of 39), neutropenia (29 [74%]), and febrile neutropenia (eight [21%]). Four non-treatment-related deaths occurred on the study drugs due to sepsis (n=2), lung infection (n=1), and undetermined cause (n=1). The median follow-up time was 10·8 months (IQR 5·6-16·4). The overall response rate was 95% (95% CI 83-99; 37/39). 19 (49%) patients proceeded to hematopoietic stem-cell transplantation. INTERPRETATION:This early analysis suggests that the combination of oral decitabine plus cedazuridine with venetoclax for higher-risk-myelodysplastic syndromes and chronic myelomonocytic leukaemia is safe in most patients, with encouraging activity. Longer follow-up will be needed to confirm these data. FUNDING:MD Anderson Cancer Center, MDS/AML Moon Shot, Genentech/AbbVie, and Astex Pharmaceuticals.

Acute myeloid leukemia (AML) is a highly aggressive hematologic malignancy with a high relapse rate primarily due to acquired therapy resistance. Accumulating evidence has shown that ferroptosis, an iron-dependent non-apoptotic form of cell death characterized by lipid peroxidation within cellular membranes, is a potential therapeutic strategy in AML that could overcome the therapy resistance. Polyunsaturated fatty acid-phospholipids (PUFA-PLs) are crucial components of cellular membranes, while particularly susceptible to peroxidation, thus could prime cancer cells to ferroptosis. Cellular PUFAs can be conjugated with Coenzyme A (CoA) for PUFA-PL synthesis, converted to acyl-CoA for energy production through β-oxidation or stored in lipid droplets (LDs) as PUFA-triglycerides (TAGs). LDs serve as the dynamic organelles for storing neutral lipids, composed primarily of TAGs and cholesteryl esters, and regulating lipid flux to meet the needs of metabolism and lipid homeostasis. Therefore, LD biogenesis conditionally buffers PUFA in TAGs, and reduces lipid peroxidation and subsequent ferroptosis. Diacylglycerol acyltransferases (DGATs), DGAT1 and DGAT2, mediate the final and rate-limiting step in TAG synthesis that drives LD biogenesis, playing critical roles in cellular lipid metabolism and homeostasis. We first confirmed that the exogenous supplement of PUFA (Linoleic acid) significantly enhanced ferroptosis in AML cells treated with the GPX4 inhibitor ML210, while monounsaturated fatty acid (MUFA, Oleic acid) suppressed GPX4 inhibition-induced ferroptosis, as expected. However, interestingly, the lipidomics analysis revealed increased levels of PUFA-TAG species in AML cells when GPX4 is inhibited. In addition, GPX4 inhibition increased cellular LD mass. Given that TAG species are the major components in LDs, we hypothesized that PUFAs are sequestered in LDs as an adaptive mechanism to mitigate oxidative lipid stress upon ferroptosis. Further analysis of lipidomics showed increased levels not only in oxidized PUFA-PL species but also in oxidized PUFA-TAG species. This implies that TAG in LDs may also scavenge the oxidized PUFA, not only unoxidized PUFA, to mitigate ferroptosis. To test the hypothesis, this study explores the potential of targeting DGATs to disrupt LD formation and enhance ferroptosis in AML cells. Supporting this notion, we demonstrated that DGAT1 inhibition by A922500 attenuated the LD formation, in contrast to negligible effects by DGAT2 inhibition. Notably, DGAT1 inhibition significantly enhanced the GPX4 inhibition-induced ferroptosis. The combination of DGAT1 inhibition with Docosahexaenoic acid (DHA), a widely used food supplement belonging to the PUFA family, further improved the ferroptotic cell death. To further validate the roles of DGAT-mediated LD formation in sensitizing AML cells to ferroptosis, we generated DGAT1 and DGAT2 stable knockout (KO) lines in OCI-AML3 and MOLM13 cells by CRISPR-Cas9. Fluorescence microscopy confirmed the obvious reduction of LDs in DGAT1-deficient AML cells. In line with the pharmacological inhibition results, DGAT1 KO increased ferroptotic cell death, while not significantly affected by DGAT2 KO, perhaps reflecting their differential functions in LD biogenesis. In summary, our research demonstrates that inhibition of LD biogenesis by selective DGAT1 inhibition and supplemental dietary PUFA synergistically enhances ferroptosis in AML cells. These findings suggest a novel therapeutic strategy for AML by targeting lipid metabolism to potentiate ferroptosis. Further investigation into the molecular underpinnings of the synergy, in vivo validations, and the development of specific DGAT inhibitors will be critical for translating these findings into clinical applications.

Background: Acquired resistance to apoptosis-targeted therapies is a major mechanism underlying relapsed and refractory acute myeloid leukemia (AML). Exploring non-apoptotic regulated cell death (RCD) could circumvent apoptosis-related resistance, therefore, has the potential of improving outcomes in AML. Ferroptosis is a form of RCD driven by iron-dependent lipid peroxidation (LP), which is negatively regulated by GPX4. We recently reported that ferroptosis in AML uniquely relies on the LP of mitochondrial membranes, which we term “mitochondrial ferroptosis” (Leukemia;38:729-740, 2024). This result prompted us to investigate the potential interrelationship between mitochondrial ferroptosis and apoptosis pathways in AML. Here, we evaluated the combinatorial anti-AML efficacy of dual induction of RCD, by inducing apoptosis and ferroptosis, and investigated the molecular interactions. Results: We evaluated the anti-AML efficacy of dual induction of apoptosis and ferroptosis utilizing venetoclax (Ven) and ML210, a specific GPX4 inhibitor, as apoptosis and ferroptosis inducers, respectively. Treatment with ML210 in combination with Ven induced synergistic cell death in various AML cell lines: the combination index was 0.71, 0.53, and 0.57 in OCI-AML3, MOLM13, and MV4;11 cells, respectively. This synergism was also recapitulated in doxycycline-inducible GPX4 knockdown cells, supporting the on-target effects of pharmacologic GPX4 inhibition. Interestingly, Ven-resistant (Ven-R) cell lines exhibited more prominent synergism compared to the parental Ven-sensitive cells. Furthermore, the synergism was also observed in AML stem/progenitor cells (CD34+CD38-) obtained from Ven-R as well as Ven-naïve AML patients. As starting point for our investigation of interactions between mitochondrial ferroptosis and apoptosis, we observed that ferroptosis of AML cells induces cytochrome c (CytC) release from mitochondria, which is a hallmark of apoptosis. Importantly, the ferroptotic CytC release was mitochondrial lipid peroxidation-dependent but BAX/BAK-independent, suggesting a mechanistic divergence from apoptotic BAX/BAK-dependent CytC release by Ven. Since metabolic rewiring to fatty acid metabolism underlies Ven-R in AML, we hypothesized that Ven affects lipid metabolism and may further sensitize AML cells to ferroptosis. Indeed, while Ven alone only slightly induced LP in AML cells, the combination of ML210 and Ven significantly enhanced LP compared to ML210 alone. The synergistic effect was attenuated by ferrostatin-1, a selective ferroptosis inhibitor. Notably, a mitochondria-targeted antioxidant, MitoTEMPO, completely blocked LP and cell death induction by the combinatorial treatment. Consistently, mass-spectrometry-based mitochondrial proteome analysis suggested that Ven reduced glutathione reductase, which is critical for the function of GPX4 by catalyzing the conversion of oxidized glutathione (GSSG) to its reduced form GSH. These results suggest that mitochondrial ROS accumulation is an essential trigger for the synergistic anti-AML effects. Furthermore, pretreatment with Ven followed by ML210 significantly increased LP and cell death compared to concomitant treatment or pretreatment with ML210 followed by Ven. This indicates a non-apoptotic role of Ven in priming AML cells into a pro-ferroptotic state. Conclusion: Mitochondrial ferroptosis exhibits molecular interactions with apoptosis pathways in AML: ferroptosis induces CytC release in a BAX/BAK-independent manner, while apoptosis sensitizes AML cells to LP possibly by affecting mitochondrial lipid and oxidative metabolism. Targeting the ferroptosis-apoptosis interactions could evolved into a novel therapeutic strategy for AML.

Resistance to apoptosis in acute myeloid leukemia (AML) cells causes refractory or relapsed disease, associated with dismal clinical outcomes. Ferroptosis, a mode of non-apoptotic cell death triggered by iron-dependent lipid peroxidation, has been investigated as potential therapeutic modality against therapy-resistant cancers, but our knowledge of its role in AML is limited. We investigated ferroptosis in AML cells and identified its mitochondrial regulation as a therapeutic vulnerability. GPX4 knockdown induced ferroptosis in AML cells, accompanied with characteristic mitochondrial lipid peroxidation, exerting anti-AML effects in vitro and in vivo. Electron transport chains (ETC) are primary sources of coenzyme Q10 (CoQ) recycling for its function of anti–lipid peroxidation in mitochondria. We found that the mitochondria-specific CoQ potently inhibited GPX4 inhibition–mediated ferroptosis, suggesting that mitochondrial lipid redox regulates ferroptosis in AML cells. Consistently, Rho0 cells, which lack functional ETC, were more sensitive to GPX4 inhibition–mediated mitochondrial lipid peroxidation and ferroptosis than control cells. Furthermore, degradation of ETC through hyperactivation of a mitochondrial protease, caseinolytic protease P (ClpP), synergistically enhanced the anti-AML effects of GPX4 inhibition. Collectively, our findings indicate that in AML cells, GPX4 inhibition induces ferroptosis, which is regulated by mitochondrial lipid redox and ETC.

Background: Most patients with acute myeloid leukemia (AML) experience relapse after initial remission, especially when unable to complete standard consolidation strategies such as high-dose cytarabine or allogeneic stem cell transplantation (SCT). Ideal maintenance therapy consists of the prolonged administration of effective lower intensity agents with the goal of delaying or preventing relapse. Oral azacitidine (QUAZAR AML-001) and 3-day IV decitabine (ECOG ACRIN E2906) have demonstrated efficacy as maintenance. In this phase 1b multi-arm study (NCT05010772), we evaluated the combination of ASTX727 (decitabine/cedazuridine), an oral formulation of decitabine with equivalent AUC to IV decitabine, plus physician choice of an oral targeted agent as personalized maintenance therapy in AML. Methods: This study enrolled patients ≥ 18 years with AML in first complete remission (CR) or CR with incomplete blood count recovery (CRi) unable to complete standard therapy and not immediately eligible for SCT. Patients who had received intensive induction (at least 2 cycles of intermediate/high dose cytarabine-based chemotherapy) were enrolled in cohort 1. Patients who had received at least 3 cycles of low-intensity therapy (hypomethylating agent or low-dose cytarabine-based regimens) were eligible for cohort 2. ECOG ≤ 3 and adequate bone marrow function (neutrophils > 0.5 x 109/L, and platelets > 50 x 109/L) were required. All patients received ASTX727 35/100 mg PO on D1-3. Patients were assigned to one of 5 arms per physician decision. Arm A consisted of ASTX727 alone. A second agent was added for the remaining arms (Arm B: venetoclax 400 mg (adjusted for CYP3A4 inhibitor use) PO on D1-5; Arm C: gilteritinib 120 mg PO on D1-28; Arm D: enasidenib 100 mg PO on D1-28; Arm E: ivosidenib 500 mg PO on D1-28). Each arm included an initial safety lead-in cohort using a 3+3 dose de-escalation design in the event of dose limiting toxicity (DLT). Cycles were 28 days and therapy continued for up to 24 cycles. The primary objective was safety/tolerability. Secondary objectives included overall survival (OS) and relapse-free survival (RFS) starting from the time of enrollment. Time-to-event endpoints were censored at the time of SCT. Results: 31 patients have been enrolled (15 in cohort 1, 16 in cohort 2). 4 patients have been enrolled in arm A, 25 in arm B, 1 in arm C, and 1 in arm D. The median follow-up time is 15.6 m. The median age for the full cohort was 68 years (33-83). ELN 2022 risk was favorable in 13 patients (42%), intermediate in 3 (10%), and adverse in 15 (48%). TP53 mutations were identified in 1 (3%) patient and complex cytogenetics in 3 (10%) patients. 4 (13%) patients had antecedent MDS/MPN and 3 (10%) had therapy-related AML. 9 (29%) patients had detectable measurable residual disease (MRD) at the time of enrollment. The median number of cycles given is 5 (1-24). No DLT's have been noted in cycle 1. The most common grade 3/4 adverse events were neutropenia (100%), leukopenia (100%), thrombocytopenia (83%), and anemia (57%). Neutropenic fever occurred in 20% of patients. 1 (3%) patient went off protocol due to toxicity (prolonged thrombocytopenia after 6 cycles) and 1 death occurred while on maintenance. For the full cohort, the median OS is not reached (NR; 82% at 1 year) and the median RFS is 22.4 months (m) (59% at 1 year). In cohort 1 (intensive induction), the median OS is NR (100% at 1 year) and the median RFS is NR (76% at 1 year). In cohort 2 (low-intensity induction), the median OS is NR (69% at 1 year) and the median RFS is 11.7 m (43% at 1 year). In arm A (ASTX727 alone), the median OS is NR (100% at 1 year) and the median RFS is 16.8 m (50% at 1 year). In arm B (ASTX727 plus venetoclax), the median OS is NR (78% at 1 year) and the median RFS is NR (65% at 1 year). Of the 9 patients with detectable MRD at enrollment, 2 (22%) became MRD-negative while on maintenance. The median OS was 11.3 m in the MRD-positive patients versus NR in the MRD-negative patients (n=22; p < 0.01). The median RFS was 6.6 m in the MRD-positive patients versus NR in the MRD-negative patients (p < 0.001). Conclusions: Combination targeted oral maintenance is feasible in AML and demonstrates encouraging RFS. Myelosuppression is universal in subsequent cycles. Prophylactic anti-infectives are encouraged. Further enrollment exploring dose/schedule modifications of ASTX727 on subsequent cycles and longer follow-up are required to confirm the efficacy of these regimens.

Introduction: Secondary myeloid neoplasms (MN) are a recognized risk following chimeric antigen receptor (CAR) T-cell therapy. Prior studies are limited to low-resolution adverse event reporting or small case series. We provide a comprehensive account of MN characteristics and outcomes following CAR T. Methods: We reviewed our institutional experience with consecutive MN cases diagnosed after CAR T therapy for other hematologic malignancies since 2015. All preceding lines of therapy (LoT), including those administered before histologic transformation, were documented. Autologous stem cell transplant (autoSCT) was counted as a unique LoT. We compared available bone marrow samples from the immediate pre-CAR T period with those from MN diagnosis to identify clonal expansion. Results: Of 1,484 patients treated with immune effector cells at our center, 37 (2.5%) received a subsequent MN diagnosis. Patients primarily received CD19- (81%) or BCMA- (16%) targeting CAR T products, all with fludarabine and cyclophosphamide lymphodepletion, at a median of 65 years of age (IQR 60-69) after a median of 5 (range 1-11) prior LoT for non-Hodgkin B-cell lymphoma or multiple myeloma (MM). Forty-three percent had undergone prior autoSCT with BEAM or melphalan conditioning. All patients had prior exposure to alkylating agents, and 41% had previous exposure to lenalidomide (len). Complete response rate post initial CAR T was 87%. Of the 11 patients who required additional therapy for the primary malignancy, 2 received a second CAR T infusion. Among the patients (28 with MDS, 1 with CMML, 8 with AML) who developed a MN, 70% were male. At the time of MN diagnosis, 95% of patients had their primary malignancy in remission. Median baseline bone marrow blast percentage was 6% (IQR 2-11) and 46% of cases had ≥ 2% ring sideroblasts. Cytogenetic risk was chiefly adverse: 46% complex karyotype, 62% deletion 7/7q, 14% MECOM rearranged and 3% KMT2A rearranged. While no patients had pure erythroid leukemia, 18 had a multi-hit TP53 status per International Consensus Classification 2022 criteria. The MN was diagnosed a median of 16.1 months (IQR 8.8-24.4) following CAR T infusion, and a median of 5.5 years, 5.1 years, and 5.7 years after first autoSCT, alkylating agent, and len exposures, respectively. One third of patients received only supportive care and 65% were treated with hypomethylating agents. Most (80%) patients had no response to initial therapy. Of the 6 patients who underwent allogeneic stem cell transplant (alloSCT), 3 died from infection or graft-versus-host disease. Of the 3 patients who experienced a durable remission post alloSCT, 2 had a wild-type TP53 status. Median overall survival (mOS) after MN diagnosis was 9.0 months (95% CI 6.1, 16.2). The majority (89%) of deaths were from MN-related complications. No statistically significant associations were identified between post MN survival and sex, age ≥ 65 years at CAR T infusion, prior autoSCT, ≥ 3 LoT pre-CAR T or TP53 status. There was an observed survival benefit with len exposure: mOS 14.3 months (95% CI 9.0, NE) with versus (vs) 5.1 months (95% CI 2.3, 16.1) without prior len exposure (p = 0.029). This association persisted when MM cases were excluded (p = 0.005). A trend towards improved survival was observed in alloSCT recipients: median OS 14.3 months with vs 7.5 months without transplant, although it did not reach statistical significance. All 4 patients with documented TP53 mutation(s) (variant allele frequency [VAF] detection limit 0.3 to 2%) within 12 months prior to CAR T, and without active marrow involvement from the primary malignancy, demonstrated clonal expansion (relative VAF increase ≥ 50%), often accompanied by a second TP53 hit. Retrospective TP53 sequencing and T-cell profiling in available marrow samples are being explored. Conclusions: Our incidence of MN following CAR T is reassuringly rare and comparable to an incidence of 1.4% from publicly available FDA data. The 5-year latency from pre-CAR T therapies to MN diagnosis aligns with historical therapy-related MN latencies, underscoring their role in malignant transformation. The shorter latency after CAR T therapy is confounded by lead-time bias but raises the question of whether CAR T-induced immune dysregulation amplifies clonal selection. Screening for TP53 mutations before CAR T administration may inform MN risk in a patient population now often cured of their primary malignancy.

Background: Isocitrate dehydrogenase (IDH) mutations occur in ~20% of patients (pts) with acute myeloid leukemia (AML). Long-term follow up of the VIALE-A trial demonstrated a median overall survival (mOS) of 10.2 and 27.5 months for those with IDH1 and IDH2 mutations, respectively, with composite complete remission (CRc) rates of 66.7% and 86%. Targeted IDH inhibitors (IDHi) such as ivosidenib (IVO) and enasidenib (ENA) are also effective, either as single agents or in combination with azacitidine (AZA). All these regimens are effective, yet not curative, and when relapse occurs it is frequently due to the emergence or expansion of signaling mutations. “Triplet” regimens may more optimally prevent common resistance mechanisms by targeting multiple pathways simultaneously. In this pooled analysis, we report the clinical outcomes and patterns of relapse among pts with newly diagnosed (ND) IDH mutant AML who are not eligible for intensive chemotherapy treated with frontline triplet regimens containing a HMA + venetoclax (VEN) + IDHi. Methods: Adults with ND IDH-mutated AML treated on IDHi “triplet” trials at our institution were included in this pooled analysis (NCT03471260 and NCT04774393). Pts received either frontline AZA + VEN + IVO or oral Decitabine/Cedazuridine (ASTX727) + VEN + IVO/ENA (arms for IDH1 or IDH2 mutant disease, respectively). OS, event free survival (EFS), and duration of remission (DOR) were assessed by Kaplan-Meier method, with EFS defined by lack of response, relapse, or death. CRc was defined as CR + CRi + CRh and overall response rate (ORR) as CR + CRi + CRh + PR + MLFS within five cycles. Results: From October 2019 to March 2024, 50 pts with ND IDH-mutated AML were enrolled; 12 pts (24%) received AZA + VEN + IVO and 38 (76%) received ASTX727 + VEN + IDHi. Median age was 71 years (range: 62 - 87) and ELN 2022 risk was favorable for 10 (20%), intermediate for 4 (8%), and adverse for 36 (72%). Twelve pts (24%) had treated secondary AML (tsAML) with prior HMA ± VEN from antecedent MDS or MPN. NPM1 mutations were present in 12 (24%), FLT3-ITD in 3 (6%), K/NRAS in 10 (20%), and TP53 in 3 (6%). At a median (m) follow up of 18.1 months, the mOS and mEFS have not been reached with a 2-year OS of 82% (95% CI: 70 - 95%), 2-year EFS of 63% (49 - 82%), and mDOR of 27 months. The ORR was 96% (48/50) with CRc of 92% (36/40). Measurable residual disease negativity by flow cytometry was achieved in 78% (36/46) of evaluable pts. Twelve responding pts (24%) transitioned to stem cell transplantation (SCT). For pts who did not undergo SCT, the median number of completed treatment cycles is 6 (IQR: 3 - 15) with 20/36 (56%) of responding pts still on active treatment. There was no difference in outcomes between pts with IDH1 or IDH2 mutations, treatment with AZA + VEN + IVO versus ASTX727 + VEN + IDHi, or in pts with baseline signaling mutations. However, pts with tsAML experienced significantly worse outcomes compared to non-tsAML with a CRc rate of 75% (9/12) versus 98% (37/38) respectively (p=0.038). tsAML pts experienced mOS of 11 months, mEFS of 7.6 months, and mDOR of 8.5 months; treatment naïve participants experienced a 2-year OS of 94% (95% CI: 84 - 100%) and 92% (95% CI: 79 - 100%) for IDH1 and IDH2 mutant AML, respectively. Ten of the 48 responding pts relapsed (21%). Five of the 6 pts with IDH1 mutations at baseline had no detectable IDH1 mutation at relapse, similar to 2 of the 4 pts with IDH2 mutations. Only 2 of 9 tested pts developed emergent signaling mutations: one gained a FLT3-ITD mutation along with new ETNK1, GNAS, SETBP1, and TET2 mutations, while the other with baseline NPM1, IDH2, and NRAS mutations gained a KRAS and a second NRAS mutation. Overall the triplet regimens were well tolerated. 30-day and 60-day mortality were 0% and 2% (1/50), respectively. Grade 3 or higher non-hematologic adverse events were experienced in 10% of pts (5/50), including 2 cases of indirect hyperbilirubinemia and 1 case each of mucositis, QTc prolongation, and differentiation syndrome. Conclusions: Pts with IDH mutant AML experience excellent outcomes with frontline HMA + VEN + IDHi triplets. There do not appear to be any recurrent mutational patterns at relapse on IDHi triplet regimens, with most IDH1 mutant pts and 50% of IDH2 mutant pts relapsing with an IDH wildtype clone. Given these promising frontline results, prospective studies comparing triplet versus doublet regimens for IDH mutant AML are warranted. Enrollment on both HMA + VEN + IDHi triplet trials is ongoing.

Background: Patients with treatment-naïve (TN) chronic lymphocytic leukemia (CLL) often receive treatment with BTK inhibitors (BTKi) with or without anti-CD20 monoclonal antibody. At the present time, it is recommended that therapy with BTKi is continued long-term, however prolonged treatment with BTKi can be complicated by side effects and/or development of resistance. As an alternative, we developed a time-limited combination therapy of acalabrutinib and obinutuzumab that incorporates treatment discontinuation after 24 cycles of therapy. We report results from the first 37 patients with TN CLL enrolled in this trial (NCT04505254). Methods: Patients with TN CLL and indication for therapy per iwCLL criteria receive acalabrutinib, 100 mg BID for 24 cycles (each cycle is 28 days), combined with monthly obinutuzumab infusions for 6 months, starting in cycle 3. Patients who achieve complete remission (CR) after 6 cycles of combination therapy continue with acalabrutinib as monotherapy. Patients with a PR or SD receive 6 additional cycles of the combination therapy (cycles 9 - 14) before transitioning to acalabrutinib as monotherapy. Treatment is discontinued after 24 cycles in all patients. After treatment discontinuation, patients transition to observation and can resume therapy if they relapse and meet iwCLL criteria for starting salvage therapy. The primary objective is to determine the durability of treatment-free remissions after 24 cycles of therapy. Secondary objectives include assessing the efficacy of re-treatment, and determining factors associated with prolonged remission. Correlative studies of T-cell compartment during treatment are ongoing. Results: 37 patients with the median age of 68 years (range, 40 - 83 years) have been enrolled and 23 are evaluable for a response. About 5% have del(17p) or TP53 mutation, 38% have unmutated IgHV and 51% have advance stage disease (Rai stage III/IV). After a median follow-up of 14 months, 36 patients are alive (97%), one patient died due to complications from bacterial pneumonia during cycle 3; 33 patients remain on study (89%). Two patients were taken off study due to recurrent infections (cholecystitis, cellulitis), and one due to loss of health insurance. The estimated two-year PFS and OS are 96.7%. In this ongoing study, the overall response rate is 100% in evaluable patients, with CR observed in 12 patients (52%) and PR in 11 patients (48%) as best response. CR rate after 24 cycles of therapy is 62%. The median levels of measurable residual disease (MRD) in the bone marrow (BM) evaluated by flow cytometry decreased from 84.30% at baseline (median, range, 36.10 - 94.70%) to 0.12% (range, 0 - 3.60%) after 24 cycles with 4 patients (31%) achieving undetectable MRD in the BM. At this time, 13 patients have already completed 24 cycles of therapy and discontinued treatment. With a median follow-up of 9 months after treatment discontinuation, 10 patients remain in remission and 3 patients experienced disease relapse, two of them requiring retreatment. Conclusions: Time-limited therapy with acalabrutinib and obinutuzumab induces remissions in patients with TN CLL with 62% of patients achieving a CR. After a median follow-up of 9 months after therapy discontinuation, 10/13 (77%) of patients remain in remission. Ongoing correlative studies and longer follow-up will define the patient characteristics that are associated with long remissions after time-limited therapy with acalabrutinib and obinutuzumab. This may help to identify patients for whom this approach could become an alternative to continuous BTK inhibitor therapy.

To evaluate the outcomes of patients with 3q26.2/MECOM-rearranged chronic myeloid leukemia (CML). We reviewed consecutive adult patients with 3q26.2/MECOM-rearranged CML between January 1, 1998 and February 16, 2023. Rearrangements of 3q26.2/MECOM were confirmed by conventional cytogenetics, and fluorescence in situ hybridization starting in 2015. We identified 55 patients with MECOM-rearranged CML, including 23 in chronic phase (CP) or accelerated phase (AP) and 32 in blast phase (BP). Nine patients (16

Introduction: Combined treatment withcovalent BTK-inhibitor (cBTKi), such as ibrutinib, acalabrutinib, or zanubrutinib with BCL2-inhibitor, venetoclax, +/- CD20 monoclonal antibody obinutuzumab showed high rates of undetectable MRD (U-MRD4, 10-4 sensitivity) remission in patients (pts) with CLL (Jain, NEJM 2019; Munir NEJM 2023; Wierda, JCO 2021; Kater, NEJM Evidence 2022). We report results of time-limited, combination therapy with non-covalent BTKi, pirtobrutinib with venetoclax and obinutuzumab as first-line treatment for pts with CLL (NCT05536349). Methods: Pts with previously untreated CLL meeting iwCLL treatment criteria were enrolled. Pts received pirtobrutinib 200mg daily starting Cycle 1 Day 1 (C1D1) continuously until end of C13. Obinutuzumab was given as standard 6 cycles starting C1D1. Venetoclax standard ramp-up was initiated C2D1 to the target dose of 400mg daily and then continued until end of C13. Each cycle is 28 days. Response evaluations (iwCLL 2018 criteria) were done by imaging and bone marrow (BM) assessment at the end of C7 (6-month of the triplet combination) and C13. MRD was assessed by ClonoSEQ next-generation sequencing in both blood and BM at the end of C7 and C13. Pts with detectable MRD5 (≥10-5 in either blood or BM) at the end of C13 can continue pirtobrutinib and venetoclax for another 12 cycles per protocol. All pts, once off therapy, are monitored by blood MRD by NGS every 3 months for the first 12 months off therapy, and then every 6 months. Results: Between February 2023 and June 2024, a total of 74 pts were enrolled. Median age is 64 years (range, 38-79 years). 77% had IGHV-unmutated CLL. 12% had del(17p)/TP53 mutation. 2 pts came off trial (1 pt in C3 for treatment of newly diagnosed head/neck cancer; 1 pt in C1 due to noncompliance with study requirements); the remaining 72 pts continue on the trial. The median follow-up is 10.3 months (range, 1.1-17.5 months). At the end of C7, among the 43 pts who reached this time-point, BM and blood NGS showed U-MRD6 (10-6 sensitivity) in 28/43 (65%) and 34/43 (79%), respectively. The corresponding U-MRD4 rates at end of C7 were 39/43 (91%) in BM and 40/43 (93%) in blood. At the end of C13, among the 27 pts who reached this time-point, BM and blood NGS showed U-MRD6 in 22/27 (81%) and 24/27 (89%), respectively. The corresponding U-MRD4 rates at end of C13 were 26/27 (96%) in BM and 27/27 (100%) in blood. Grade 3-4 neutropenia and thrombocytopenia occurred in 58% and 18% pts, respectively. No pt has progressed/died. Of the 27 pts who completed C13, 24 discontinued all therapy and are in post treatment follow-up; the remaining 3 pts are continuing additional 12 cycles of pirtobrutinib and venetoclax per protocol due to detectable MRD5 at the end of C13. Conclusions: We report results of combined pirtobrutinib, venetoclax, and obinutuzumab in pts with previously untreated CLL. We observed a very high rate of BM U-MRD6 at 6-months and 12-months of combined treatment. Adverse event profile was similar to what was noted in previous studies with these agents. Updated data will be presented.

Supplementary Methods, Tables, and References

Detailed results in apoptosis assay of primary AML cells treated with DS-3032b.

Supplemental Methods Table S1. Growth inhibition by DS-3032b and nutlin-3a in tumor cell lines with different p53 status. Table S2. Percent tumor growth inhibition (%TGI ) values at day 20 in SJSA-1 xenograft mice treated with DS-3032a. Table S3. GI50 values of cancer cell lines treated with DS-3032b. Table S4. Clinical information of the 41 samples Table S5. Confusion matrix of predicted and actual sensitivity of primary AML cells to DS-3032b according to TP53 mutation status. Table S6. The cut-off values used for definining sensitivity/resistance to each drug in the different phases of the signature development and validation process. Table S7. Prediction of sensitivity by 175-gene signature scoring and actual results in patient-derived xenograft models of various tumors treated with DS-3032a. Table S8. Confusion matrix of predicted and actual sensitivity of primary AML cells to DS-3032b using previously reported 4-gene signatures. Table S9. The top-ranked p53-inducible genes in the 175-gene signature. Table S10. Cancer type and TP53 mutational frequency. Table S11. Referenced 32 genes. Figure S1. Effects of DS-3032b as an MDM2 inhibitor in vitro and in vivo. Figure S2. Effects of MDM2 inhibitors (DS-3032b, DS-5272 and nutlin-3a) in OncoPanel cell lines. Figure S3. Prediction of sensitivity of tumor cells to MDM2 inhibition using the 175 gene signature.