Venetoclax In Acute Myeloid Leukemia Research Articles

Abstract 5852: Tumor gene expression patterns affecting response to BCl-2 inhibitor venetoclax in acute myeloid leukemia

Abstract Background: Venetoclax (VEN), a B-cell lymphoma-2 (BCL-2) inhibitor, is an important drug in acute myeloid leukemia (AML) treatment regimens. However, resistance to VEN-based therapy is a common challenge in AML management. Some factors contributing to resistance include dysregulation of BCL-2 family proteins, mutations in the TP53 gene, activating kinase mutations, and oxidative phosphorylation activity. Despite these insights, other mechanisms of resistance remain elusive. We aim to analyze tumor gene expression patterns associated with treatment response in patients who received VEN-based therapy. Methods: Molecular and clinical data of the patients were obtained as part of the Oncology Research Information Exchange Network (ORIEN) Avatar dataset. RNA-Seq data from diagnostic Bone Marrow were processed to remove batch effects, and differential expression analysis was performed using DESeq2, while correcting for age at sample collection and sex of patients. Results: We included 16 individuals treated with VEN-based therapy. Median age at the time of specimen collection was 65 years (interquartile range, 58-69), 9 (56%) male, 11 (69%) had active disease despite treatment with VEN. VEN-based therapy was used as 1st line, 2nd line, 3rd line, 4th line or more in 3, 4, 4, 2, and 3 patients, respectively. We found a total of 332 genes differentially expressed (fdr<0.05 and abs(log2FoldChange)>2.0) among patients having no evidence of active disease and those with no active disease at the time of last contact. Among those genes, we found HOXA7 and PRAME of particular interest. HOXA7 has been previously implicated in Bromodomain and Extraterminal Domain Inhibitors (BETi) inhibitor resistance in AML. This makes HOXA7 a potentially universal mechanism of chemotherapy resistance in AML. PRAME has been associated with leukemia cell survival, and its knockdown has been shown to induce apoptosis in K562 cells, which is only expressed in AML cells but not in normal hematopoietic cells. Pathway analysis found that the top two enriched pathways for the differentially expressed genes were MET activates PTK2 signaling (R-HSA-8874081) and Metabolism of Angiotensinogen to Angiotensins(R-HSA-2022377). Conclusions: This study highlights some molecular mechanisms that give rise to AML resistance to VEN. Our results indicate that HOXA7 may potentially be a universal mechanism of chemotherapy resistance not limited to VEN-based therapy. Similarly, pathways enriched in resistant cells could be targeted to prevent or treat resistance to VEN-based therapy. These and other findings may help us identify resistance mechanisms in all AML cases vs those specific to VEN-based therapy, allowing the development of treatment strategies based on treatment mechanism. Also, analysis of these mechanisms can aid therapeutic decision-making and the development of effective treatment strategies to improve outcomes in AML. Citation Format: Juan Antonio Raygoza Garay, Seongseok Yun, Afaf Osman, Reshma Ramlal, David Feith, Michelle Churchman, Prajwal Dhakal. Tumor gene expression patterns affecting response to BCl-2 inhibitor venetoclax in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5852.

Abstract 6564: SOS1 inhibitor treatment improves response to Venetoclax in acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. First-line treatment of AML patients consists primarily of chemotherapy, sometimes combined with a targeted therapy. However, elderly patients unfit for chemotherapy receive venetoclax, a BCL2 inhibitor, combined with azacitadine or decitabine. While responses in these patients are high (50-70%, with median OS of 10-17 months), around 30-50% of them do not respond and most relapse within 1-10 years. Increased MAPK pathway activity or low frequency RAS pathway mutations have been identified as key mediators of resistance to standard-of-care targeted therapies for AML, including venetoclax. Here, we found that in DepMap whole-genome CRISPR screens, leukemia cell lines, including AML, show a particular dependency on SOS1. A subsequent PRISM screen with a selective inhibitor of SOS1 confirmed these results. To validate these results in vitro, we treated 7 AML cell lines showing strongest dependency on SOS1 based on DepMap and PRISM screens, with SOS1i or a SHP2 inhibitor. We found that in 4 of 7 cell lines treatment with SOS1i or SHP2i strongly impaired proliferation. In 3 of 4 responding cell lines, the anti-proliferative effect with either SOS1i or SHP2i was stronger than azacytidine single agent treatment. We next treated 29 primary ex vivo cultures of AML with SOS1i and found that the treatment induced strong or intermediate anti-proliferative effect in 21 of 29 models (72%). In two AML PDX models, treatment with SOS1i as a single agent resulted in approximately 25% reduction of human CD45+ AML blast cells in both models. Venetoclax treatment led to approximately 75% decrease in AML blasts, while the combined venetoclax and SOS1i treatment resulted in near complete eradication of the disease. Annexin V staining of AML monoblasts demonstrated that this decrease was due to an increase in apoptosis in the combination groups. In summary, we demonstrate that SOS1 inhibitor treatment may be used to improve responses to venetoclax in AML patients. Citation Format: Kaja Kostyrko, Melanie Hinkel, Matthew G. Rees, Melissa M. Ronan, Jennifer A. Roth, Daniel Gerlach, Irene Waizenegger. SOS1 inhibitor treatment improves response to Venetoclax in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6564.

Mechanisms of resistance to venetoclax in hematologic malignancies.

Venetoclax, a BH3 mimetic, is a novel targeted anti-cancer drug with a unique mechanism of action leading to the execution of apoptosis through inhibition of the Bcl-2 protein. The development of venetoclax has revolutionized the treatment paradigm of several hematologic malignancies, including treatment-naïve and relapsed or refractory chronic lymphocytic leukemia (CLL) as well as acute myeloid leukemia (AML) in unfit patients. However, despite the high effectiveness of venetoclax in these diseases, some patients, as in the case with other targeted therapies, develop primary or secondary resistance to the drug. Various mechanisms contributing to the resistance to venetoclax have been elucidated, including selection of mutations in the BCL-2 binding groove which decrease affinity to venetoclax, or compensatory overexpression of anti-apoptotic proteins such as MCL-1. Moreover, alterations in cell metabolism and signaling pathways like MAPK or ERK activation have also been reported, suggesting the resistance to venetoclax is highly complex and involves multiple pathways. This review aimed to describe the mechanisms of resistance to venetoclax in AML, CLL, multiple myeloma, and other hematologic malignancies, as well as to propose a perspective to circumvent it.

The UBE2J2/UBE2K-MARCH5 ubiquitination machinery regulates apoptosis in response to venetoclax in acute myeloid leukemia

The UBE2J2/UBE2K-MARCH5 ubiquitination machinery regulates apoptosis in response to venetoclax in acute myeloid leukemia

Licochalcone A induces mitochondria-dependent apoptosis and interacts with venetoclax in acute myeloid leukemia

The management of patients with acute myeloid leukemia (AML) remains a challenge because of the complexity and heterogeneity of this malignancy. Despite the recent approval of several novel targeted drugs, resistance seems inevitable, and clinical outcomes are still suboptimal. Increasing evidence supports the use of natural plants as an important source of anti-leukemic therapeutics. Licochalcone A (LCA) is an active flavonoid isolated from the roots of licorice, Glycyrrhiza uralensis Fisch., possessing extensive anti-tumor activities. However, its effects on AML and the underlying mechanisms remain unknown. Here, we showed that LCA decreased the viability of established human AML cell lines in a dose- and time-dependent manner. LCA significantly induced mitochondrial apoptotic cell death, accompanied by the downregulation of MCL-1, upregulation of BIM, truncation of BID, and cleavage of PARP. A prominent decline in the phosphorylation of multiple critical molecules, including AKT, glycogen synthase kinase-3β (GSK3β), ERK, and P38 was observed upon LCA treatment, indicating PI3K and MAPK signals were suppressed. Both transcription and translation of c-Myc were also inhibited by LCA. In addition, LCA enhanced the cytotoxicity of the BCL-2 inhibitor venetoclax. Furthermore, the anti-survival and pro-apoptotic effects were confirmed in primary blasts from 10 patients with de novo AML. Thus, our results expand the applications of LCA, which can be regarded as a valuable agent in treating AML.

Hypomethylating Agents and Venetoclax for Acute Myeloid Leukemia Relapsed After Hematopoietic Stem Cell Transplant

BackgroundHypomethylating agent + venetoclax is an effective frontline combination for acute myeloid leukemia, but its efficacy and safety in post-allogeneic hematopoietic cell transplant (alloHCT) relapse remain underexplored. Outcomes have been poor for this population, with no standard treatment. Patients and MethodsWe retrospectively analyzed 72 Ven-naïve patients who received hypomethylating agents + venetoclax at relapse following alloHCT and aimed to evaluate the rates of complete remission with or without hematologic recovery (CR/CRi) and minimal residual disease (MRD) negativity, CR/CRi duration, and overall survival. We leveraged our larger sample to analyze the impact of cytogenetic/molecular features on the odds of CR/CRi. ResultsCR/CRi was achieved among 32 of 67 (48%) patients, and MRD negativity was recorded among 10 of 12. NPM1 and IDH 1 or 2 mutations increased the odds of CR/CRi, as did increasing time from alloHCT to relapse. Fourteen patients subsequently received donor lymphocyte infusions or a second alloHCT. Responses lasted a median of 17.8 months (95% CI, 7.2 months to not reached), and responders had a greater median overall survival of 19.7 months (95% CI, 7.6-51.5 months) compared to 2.9 months among nonresponders (95% CI, 1.8-4.4 months; log-rank P < .01). Treatment was well tolerated, but prolonged cytopenias were common and most patients required reduction in the number of venetoclax days per cycle. ConclusionThese data support the efficacy of this combination in the alloHCT relapse setting where we report responses among nearly half of patients, with possibly greater benefit for NPM1 and IDH 1/2-mutated cases. These responses can be durable and profound as evidenced by conversion to MRD negativity.

Phase 1b trial of tagraxofusp in combination with azacitidine with or without venetoclax in acute myeloid leukemia

AbstractCD123, a subunit of the interleukin-3 receptor, is expressed on ∼80% of acute myeloid leukemias (AMLs). Tagraxofusp (TAG), recombinant interleukin-3 fused to a truncated diphtheria toxin payload, is a first-in-class drug targeting CD123 approved for treatment of blastic plasmacytoid dendritic cell neoplasm. We previously found that AMLs with acquired resistance to TAG were re-sensitized by the DNA hypomethylating agent azacitidine (AZA) and that TAG-exposed cells became more dependent on the antiapoptotic molecule BCL-2. Here, we report a phase 1b study in 56 adults with CD123-positive AML or high-risk myelodysplastic syndrome (MDS), first combining TAG with AZA in AML/MDS, and subsequently TAG, AZA, and the BCL-2 inhibitor venetoclax (VEN) in AML. Adverse events with 3-day TAG dosing were as expected, without indication of increased toxicity of TAG or AZA+/−VEN in combination. The recommended phase 2 dose of TAG was 12 μg/kg/day for 3 days, with 7-day AZA +/− 21-day VEN. In an expansion cohort of 26 patients (median age 71) with previously untreated European LeukemiaNet adverse-risk AML (50% TP53 mutated), triplet TAG-AZA-VEN induced response in 69% (n=18/26; 39% complete remission [CR], 19% complete remission with incomplete count recovery [CRi], 12% morphologic leukemia-free state [MLFS]). Among 13 patients with TP53 mutations, 7/13 (54%) achieved CR/CRi/MLFS (CR = 4, CRi = 2, MLFS = 1). Twelve of 17 (71%) tested responders had no flow measurable residual disease. Median overall survival and progression-free survival were 14 months (95% CI, 9.5-NA) and 8.5 months (95% CI, 5.1-NA), respectively. In summary, TAG-AZA-VEN shows encouraging safety and activity in high-risk AML, including TP53-mutated disease, supporting further clinical development of TAG combinations. The study was registered on ClinicalTrials.gov as #NCT03113643.

Treatment Outcomes with Hypomethylating Agents and Venetoclax for AML in the Community Compared to an Academic Setting

Background: The treatment of acute myeloid leukemia (AML) in older and/or unfit patients consists of a combination of a hypomethylating agent (HMA), such as decitabine and azacitidine, with venetoclax, which can be safely administered in the ambulatory setting. In this study, we aimed to compare the outcomes of the treatment of patients with AML who received chemotherapy with venetoclax and an HMA in an ambulatory setting at an academic center or at a community practice with academic center support. Methods: This was a retrospective review of patients with de novo or relapsed/refractory AML treated with a combination of venetoclax and either decitabine or azacitidine between February 2018 and June 2023. Patients were included if they received treatment at an academic center or if they received their treatment at a community practice with ongoing consultation at an academic center. Drugs were administered per the standard of care and local practice. The primary objective was to evaluate the overall response rate (ORR). Secondary objectives were to evaluate differences in chemotherapy management between locations, potential socioeconomic disparities, and to identify adverse events that affect the dosing or administration of therapy. Results: A total of 129 patients received venetoclax plus HMA for AML for a minimum of one cycle as seen in table 1. Of these patients, 77 (60%) received treatment at an academic center, and 52 (40%) received treatment in the community setting. The average age at treatment initiation was 67 years. Disease risk was stratified according to the 2022 European LeukemiaNet (ELN) guidelines. While not statistically significant, there was a tendency for more patients at the academic center to have adverse risk disease (77.9% vs. 69.2%, p=0.364). They were also more likely to have had exposure to a previous HMA (n=10 vs. n=7, p=1) and prior AML treatment (n=20 vs. n=17, p=0.529). Additionally, patients were more likely to have been diagnosed with secondary AML in an academic setting (n=29 vs. n=18, p=0.868). In a multivariate Cox regression model, patients in the middle- and high-income groups, defined as a median household income greater than $52,000, had a 17% lower relative risk regarding survival probability than those in the lower-income group (HR=0.83, 95% CI 0.39-1.73, p=0.622). Overall, patients in the academic setting had a 33% higher relative risk regarding survival probabilities than the community-treated with covariate consideration of performance status, ELN risk, poverty status and exposure to prior-treatments (HR=1.33, 95% CI 0.78-2.26, p=0.285). The OS of all patients was 9.67 months (95% CI 8.33-12.7), with no significant difference between the groups as seen in figure 1. Additionally, the overall response rates (ORR) of the two groups were similar at 38.5% (n=20) and 35.1% (n=27) in community and academic settings, respectively. The complete remission (CR) rate in the community was 11%, while there was an increase of 18% in the academic setting. There were also increased CR rates in the academic compared to the community setting after cycle 1 (4.7 vs. 2.9%, p=0.33), cycle 2 (11.1 vs. 4.8%, p=0.858), and cycle 3 (18.2 vs. 0%, p=0.27). Adverse events in the study were similar except for increased rates of fatigue in the academic setting (76.6% vs. 55.8%, p=0.021). Only 67% of patients underwent a bone marrow biopsy after cycle 1 in the community versus 83% in the academic setting (p=0.101). Lastly, 67% of patients underwent dose reduction with the decitabine with venetoclax regimen in the community setting versus 31% in the academic setting (p=0.012). Conclusion: This study shows that the use of venetoclax plus HMA allows patients to safely receive AML treatment in the community with support from an academic center. Although there are some significant differences in management, such as increased rates of dose reduction in the community setting, the OS and ORR are similar. However, CR rates remain lower in the community setting. It is important to note that the patients in our study, who were treated at an academic center, had disease characteristics that were more resistant to treatment. Ultimately, this study identifies our efforts in creating community partnerships. Our goal is to further evaluate this partnership with quality improvement projects to bridge the gap in AML care between the community and academic practices.

Homoharringtonine Enhances the Anti-Leukemia Effect of Venetoclax in Acute Myeloid Leukemia with t(8;21)(q22;q22) Via Regulation of c-Myc/Bim Axis

Purpose: Recently, small cases reports showed that acute myeloid leukemia (AML) with translocation (8;21)(q22;q22) [t(8;21) AML] responded poorly to venetoclax (VEN) plus azacitidine (AZA) (VA) regimen. Our previous study revealed homoharringtonine (HHT) enhanced the the killing effect of VA in AML. This study went further to explore the mechanism of VEN-resistance and the synergetic effect of VEN and HHT (VH) in overcoming resistance in t(8;21) AML. Methods: Patients with/without t(8;21) AML, being treated with VA with/without HHT were included for response assessment. Synergetic effect and mechanism of VEN combined with HHT or AZA was studied in cell lines, primary cells and mice. Results: Patients with t(8;21) AML acquired significantly lower CR/CRi rate than those without, with VA regimen as frontline (1/5 vs. 56/82, P=0.027) or salvage treatment (1/10 vs. 60/127, P=0.023), while the CR/CRi rate was comparable between the two cohorts with VA plus HHT (VAH) as salvage treatment (9/13 vs. 102/152, P&amp;gt;0.05). Synergetic effect in anti-leukemia was observed in VH but not VA in t(8;21) AML cell lines, primary cells and cell line-derived xenograft mice. Transcriptome RNA-sequencing and western blot detection found c-Myc and its downstring signialing were significantly decreased with the treatment of VH but not VA as compared to the controls. In addition, VEN induced accumulation of c-Myc in t(8;21) cells. Genetic silencing or pharmacological inhibition of c-Myc restored VEN sensitivity in t(8;21) cells. Co-immunoprecipitation assay found Bim, the crucial BH3-only protein, participated in the course of VH-inducing apoptosis. Direct or indirect inhibition of c-Myc increased expression of Bim, while overexpression of c-Myc significantly inhibited the transcription of Bim in t(8;21) cells. CUT&amp;RUN assay revealed two functional sites of c-Myc binded to the promoter of Bim gene. Conclusion: Abnormal activation of c-Myc might contribute to the resistance of VEN via suppression of Bim in t(8;21) AML. HHT might enhance the killing effect of VEN by regulation of c-Myc/Bim axis. Key words: acute myeloid leukemia, t(8;21), homoharringtonine, venetoclax, c-Myc

The Impact of Delayed Venetoclax Initiation on Overall Survival in Acute Myeloid Leukemia

Introduction The use of venetoclax in acute myeloid leukemia (AML) has become routine - following FDA approval, approximately half of patients with AML have received venetoclax. However, the outcomes of the timing of venetoclax initiation remain unknown. Such outcomes are of notable importance, as it is often critical to allow time for patient optimization and to await molecular profiling - particularly as ongoing research contributes to a deeper understanding of the mechanisms of venetoclax sensitivity and resistance. A critical real-world limitation is that the turnaround times of complete molecular profiling via NGS assays are frequently 14 to 21 days. Therefore, we performed the first stratified analysis that details the impact of delays in venetoclax-based regimen initiation on overall survival. Methods We analyzed 185 patients with AML treated with venetoclax and a hypomethylating agent (azacitidine or decitabine; VEN+HMA) from January 1, 2018 to April 18, 2023 at VCU Massey Comprehensive Cancer Center. We recorded baseline patient-related and disease characteristics, including age, ECOG, Charlson comorbidity index (CCI) scores, molecular profiling and ELN 2022 cytogenetic risk, dates of regimen initiation, and survival. We individually analyzed the first-line and relapsed or refractory (RR) groups. In each group, we separated patients into three cohorts: those that started therapy in 0-7 days, 8-14 days, and 15 days and beyond. We used the D'Agostino &amp; Pearson method for normality testing and the Mann-Whitney test for between-group comparisons. We applied the Bonferroni correction if multiple comparisons were made. We analyzed survival by the Kaplan-Meier method, with significance determined by the log-rank test. The event for calculating the overall survival (OS) was the date of death. Patients were otherwise censored at the date of last contact. Results Of the 185 patients treated with venetoclax and a hypomethylating agent, 100 (54.1%) were in the first-line setting, and 85 (45.9%) had RR AML. In the first-line setting, the median time to initiation of VEN+HMA was 5 days (range: 0-69 days). Compared with the first-line setting, VEN+HMA for RR AML was started at a significantly longer time from the date of disease progression at a median of 11.5 days (range: 0-110 days, p &amp;lt; 0.0001). In the first-line setting, the median OS was significantly worse for earlier initiation of VEN+HMA at 5.8 months for the 0-7 day cohort, compared with 8.9 months for the 8-14 day cohort and 12.7 months for 15 days and beyond (p = 0.023, Figure A). Similarly, in RR AML, the median OS was also significantly worse in the 0-7 day cohort at 4.9 months - but significantly better in the 8-14 day cohort at 10.5 months compared with the 15-day and beyond cohort at 7.1 months (p = 0.027, Figure B). Discussion In the first-line setting, short delays in initiating VEN+HMA do not adversely impact survival. This approach may be preferred to optimize comorbidities and disease-related complications while awaiting the results of molecular profiling. In RR AML, delays in therapy initiation also do not appear to decrease survival - although the optimal therapeutic window appears to be shorter. Taken together, these findings are the first to suggest that with lower-intensity venetoclax-based strategies, short delays in treatment for patient optimization and comprehensive molecular profiling are associated with improved survival.

Targeting SIRT5 By Succinylating Hadha Synergizes with Venetoclax in Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is genetically diverse, driven by multiple oncogenic factors. Metabolic dysregulation is a critical vulnerability in AML cells for targeted therapy. BCL2 has been previously reported to maintain mitochondrial oxidative phosphorylation (OXPHOS) in AML cells. One of the mechanisms of action of the combination of venetoclax and azacitidine is to inhibit amino acid metabolism, leading to cell death. Although venetoclax-based treatment regimens have shown promising clinical outcomes in AML patients, treatment resistance has become a newly emerged challenge. Leukemia stem cells isolated from venetoclax-resistant patients exhibit distinctive metabolic features, including elevated nicotinamide metabolism. SIRT5 is an NAD+-dependent deacylase enzyme, and its knockout leads to elevated succinylation of various enzymes involved in OXPHOS. Hence, we hypothesize that inhibiting SIRT5 may augment venetoclax's cytotoxic effect by dampening metabolism. To further elucidate this mechanism, we employed proteome analyses of succinylation to identify specific target molecules regulated by SIRT5. Methods: To determine the effects of combination therapy on cell proliferation, apoptosis, and colony formation, AML cell lines were transduced with lentiviruses carrying either shNC or shSIRT5, or treated with NRD167 (a specific inhibitor of SIRT5), followed by venetoclax treatment. To evaluate the clearance of leukemia cells in vivo, NSG mice were transplanted with MOLM-13 cells (carrying shNC or shSIRT5) and then subjected to vehicle or venetoclax treatment (80 mg/kg/day) via oral gavage for 14 days. Weekly live animal in vivo imaging was conducted to monitor tumor engraftment and growth. The synergistic effect on the metabolic level of AML was assessed by measuring ATP, glutathione, mitochondrial superoxide, and oxygen consumption rates (OCR). Electron microscopy was employed to detect changes in cellular mitochondrial structure after SIRT5 knockdown and venetoclax utilization. Furthermore, lysine succinylation proteome analyses were performed to identify proteins and sites with altered succinylation on lysine before and after SIRT5 knockdown in MOLM-13 cells. Immunoprecipitation assays and Western blot were conducted to verify the interaction and altered succinylation levels of the relevant proteins. Results: The combination of SIRT5 inhibition with venetoclax treatment resulted in further suppression of cell proliferation, reduced colony formation, and increased apoptosis in AML cells compared to venetoclax treatment alone.The combination led to alterations in the metabolic profile of leukemia cells, increased the generation of mitochondrial reactive oxygen species (ROS), reduced intracellular glutathione levels, inhibited ATP production, and suppressed OCR. Notably, combination therapy resulted in a significant reduction in the engraftment levels of human leukemia cells in NSG mice. Mechanistically, electron microscopy revealed that combination treatment with SIRT5 knockdown increased mitochondrial damage. Lysine succinylation proteome analyses demonstrated upregulation of succinylation modification on mitochondrial trifunctional enzyme subunit α (HADHA), a protein involved in the fatty acid oxidation (FAO) pathway. Co-immunoprecipitation of SIRT5 confirmed its interaction with HADHA, and the relative succinylation level of precipitated HADHA was upregulated after SIRT5 knockdown. Moreover, the combination of trimetazidine (a specific inhibitor of HADHA) with venetoclax demonstrated a preferential reduction in cell proliferation and induction of apoptosis in AML cells. Conclusion: Targeting of SIRT5 in combination with venetoclax treatment results in synergistic cytotoxic effects in AML cells. We hypothesize that SIRT5 desuccinylase activity promotes FAO and reduces intracellular mitochondrial ROS levels by desuccinylating HADHA. Targeting SIRT5 inhibits fatty acid metabolism in AML cells, thereby enhancing the therapeutic effect of venetoclax. We will further investigate the site(s) of SIRT5 desuccinylation modification of HADHA through lysine site mutation and assess changes in HADHA enzyme activity to elucidate its regulatory mechanism.

The UBE2J2/UBE2K-MARCH5 Ubiquitination Machinery Regulates Apoptosis in Response to Venetoclax in Acute Myeloid Leukemia

Evasion of apoptosis is crucial for the growth, survival and chemoresistance of many cancer types, including acute myeloid leukemia (AML); thus, the reactivation of apoptosis can be exploited as a therapeutic approach. Apoptosis induction is mainly controlled by the balance between anti-apoptotic and pro-apoptotic BCL2 family proteins on the mitochondrial membrane. Venetoclax, a selective inhibitor antagonizing the anti-apoptotic protein BCL2, has emerged as a promising therapy in AML. Despite high response rates in combination with hypomethylating agents, however, some patients display upfront resistance, and most patients will ultimately relapse. Therefore, identification of synergistic targets for combination therapies with venetoclax is important for improving the clinical application of this drug. To systematically identify key genes that can modulate the venetoclax effect, we performed a genome-scale CRISPR-Cas9 screen in the AML cell line MV4-11. Consistent with previous reports, loss of the apoptosis effector BAX or the pro-apoptotic gene NOXA ( PMAIP1) caused venetoclax resistance, while sgRNAs targeting the anti-apoptotic genes BCLXL ( BCL2L1), BCL2L2 and BCL2A1 were significantly more depleted in venetoclax-treated cells. Furthermore, depletion of the E2 ubiquitin-conjugating enzymes, UBE2J2 and UBE2K, ranked highly among the venetoclax sensitizers. We validated that deletion of either UBE2J2 or UBE2K increased induction of apoptosis in multiple AML cell lines and patient-derived xenograft (PDX) cells upon venetoclax treatment and was deleterious as single gene perturbation in some models. Exploiting the Broad Institute Cancer DepMap dataset, which includes genome-scale CRISPR-Cas9 screens in over 1000 cancer cell lines, revealed that dependency on UBE2J2 or UBE2K significantly correlated with a dependency on the E3 ligase MARCH5. We previously identified the repression of MARCH5 as a strong inducer of apoptosis in AML. Since E2s coordinate with ubiquitin E3 ligases to execute ubiquitination, this DepMap result suggested that UBE2J2 and UBE2K serve as MARCH5 E2 partners. To test this hypothesis, we utilized NanoBiT technology, a structural complementation reporter system, to detect protein interactions between MARCH5 and the E2 candidates. LgBIT and SmBIT, two split subunits of luciferase, were fused with MARCH5 and the E2 proteins, respectively. The luminescent signal was activated upon the co-expression of LgBIT-MARCH5 with either of the SmBIT-tagged E2 proteins but not an empty vector, suggesting that MARCH5 and UBE2J2/UBE2K constitute ubiquitination machinery that regulates apoptosis in AML. We next tested the possible redundancy between the two E2s and showed that the double knockout of UBE2J2/ UBE2K further enhanced the venetoclax sensitivity. MARCH5 depletion results in increased NOXA expression, an important node in dictating venetoclax response. Several reports indicated that NOXA is also a critical downstream mediator of MARCH5. In contrast, we previously showed that MARCH5 can regulate apoptosis independently of NOXA in AML. To reassess the role of NOXA and other pro-apoptotic proteins in MARCH5-mediated apoptosis, we conducted an unbiased CRISPR rescue screen in a MARCH5-dTAG degradation system derived from PDX17-14, a complex karyotype, MLL-AF10 PDX model. BAX was the top rescuing target, emphasizing that apoptosis induction is the main mechanism accounting for the growth inhibition of MARCH5-depleted cells. However, depletion of other pro-apoptotic BCL2 members, including NOXA and BIM, did not rescue MARCH5 depletion in this screen consistent with our previously published data. Here, we confirmed that NOXA KO does not rescue MARCH5 depletion in additional AML models. Similarly, KO of UBE2J2 or UBE2K can repress AML cell growth and increase venetoclax sensitivity even in the context of NOXA KO. Our study highlights that UBE2J2 and UBE2K are two important functional partners of MARCH5 in regulating apoptosis in AML cells and can serve as additional targets for enhancing venetoclax efficacy. Unbiased screening and low-throughput target validation further emphasize that the MARCH5 ubiquitination machinery regulates apoptosis in AML cells largely in a NOXA-independent manner. Additional studies are needed to dissect the MARCH5/UBE2J2/UBE2K complex-mediated apoptosis regulation in AML.

Functional Ex-Vivo Testing Combined with Machine Learning Predicts Clinical Response to Azacitidine+Venetoclax in AML

Background: The combination of Venetoclax + hypomethylating agents such as Azacitidine (Ven/Aza) has revolutionized the treatment of Acute Myeloid Leukemia (AML). Ven/Aza is currently the standard of care for patients with newly diagnosed AML who are not candidates for intensive chemotherapy (IC), as well as a recurrent therapeutic choice for IC-relapsed/refractory patients. Despite widespread adoption, the predictive biomarkers of Ven/Aza response/resistance available today are not sufficiently conclusive (Griffioen et al. Cancers 2022). In fact, a current hot topic in the field is whether Ven/Aza can also become a frontline therapy for fit patients as an alternative to IC. Several worldwide studies are addressing this issue, including an ongoing clinical trial named “Survival of the Fittest” exploring this concept in the US (NCT04801797). An open question is whether functional ex-vivo testing could become a powerful actionable tool that overcomes limitations presented by genetic and molecular indicators. Aim &amp; Methods: In this study, we utilized an innovative ex-vivo functional platform of Patient Micro Avatars (PMAs) developed at OncoPrecision (García et al, Blood 2022 140 (Supplement 1): 13027-13028) to assess the predictive power for Ven/Aza treatment in a cohort of 11 AML patients. Patient-Derived Cells (PDCs) were obtained from the patients' PBMCs and Ven/Aza activity was assessed using high-throughput flow cytometry. An in-house Machine Learning (ML) tool was developed to identify the optimal variables that predict clinical outcome (Complete Remission: Yes/No). Clusters of predictive Response/Non-Response were defined by analyzing a cohort of &amp;gt;60 previously profiled AML patients and by leveraging the two most predictive populational features generated by the platform that better correlate with clinical outcome from &amp;gt;12,000 combinations of variables. Results: Our findings revealed differential ex-vivo activity of Ven/Aza in AML, with remarkably contrasting outcomes that defined clearly separate clusters of response (Figure - Left Panel). Such ML clustering led to 80% Overall Predictive Power and 100% Positive Predictive Value, meaning that all the patients for whom the platform predicted response achieved complete clinical remission. Moreover, by comparing the performance of other targeted therapies such as Gilteritinib, as well as IC regimens such as AraC-FaraA-IDA, side by side we were able to identify patients for whom Ven/Aza is the optimal regimen (e.g., Patient #169), and others for whom IC or Gilteritinib emerge as more promising therapeutic options (e.g., Patient #170) (Figure - Right Panel). Conclusions: This work presents a groundbreaking approach to predict Aza/Ven responders versus non-responders in AML. Combining the phenotypic outcomes of functional ex-vivo testing with ML tools presents the opportunity to more comprehensively predict response to cutting-edge therapies such as Ven/Aza than traditional genetic testing. This potential is particularly compelling for therapeutic schemes not directly associated with the acquisition of genetic mutations.

LSD1 Inhibition Synergizes with Venetoclax in Acute Myeloid Leukemia By Targeting Cellular Metabolism

Introduction: Acute myeloid leukemia (AML) is a hematological neoplasm with poor clinical outcomes. The introduction of the BCL2 inhibitor venetoclax in combination with hypomethylating agent has improved response rates in older patients with AML by targeting the metabolism of leukemic stem cells (LSCs). Resistance, however, is unfortunately still common. Thus, there remains a need to enhance the clinical efficacy of venetoclax by combining it with novel agents. In diverse AML models, lysine-specific demethylase 1 (LSD1) inhibition promotes cellular differentiation and reduces LSCs. We therefore asked if LSD1 inhibition could enhance the efficacy of venetoclax in the treatment of AML. Methods: We have studied the effect of single-agent and co-treatment with venetoclax and LSD1 inhibitor bomedemstat on cell proliferation in human AML cell lines (N=3), Hoxa9/ Meis1(H9M)- and MN1-transformed murine myeloid progenitor lines as well as primary AML patient samples (N=5). We have analyzed the effect of co-treatment with these two compounds on functional cellular bioenergetics in MOLM-13 cells using Seahorse extracellular flux analyzer and followed up by performing NMR spectrometry to study changes in metabolite abundance. Further, we have studied the efficacy of this drug combination in vivo in patient-derived xenograft (PDX) AML models (N=2). Results: Human AML cell lines showed a differential cytocidal activity at 96 hours (h) to venetoclax with MOLM-13 (IC 50 0.04 µM) being most sensitive and OCI-AML3 most resistant (IC 50 9.81 µM). A similar range (between 0.15 µM and 7.3 µM) was also observed in the primary AML samples. For bomedemstat, we observed a time-dependent response where the IC 50 decreased over time, in particular, in the most sensitive model systems. In murine H9M cells, the IC 50 decreased from 30.43 µM at 48 h to 0.034 µM at 96 h and further down to 0.0068 µM at 168 h. Combining bomedemstat and venetoclax, the IC 50 for venetoclax decreased; and synergism was observed in MOLM-13 cells, the most sensitive AML model, but no synergism was observed in venetoclax-resistant cells. Synergism was also observed in MN1 cells as well as 3 of the 5 primary AML samples studied (Table 1). To investigate the mechanism of the observed synergism, we explored the effect of these treatments on metabolic pathways using the Seahorse extracellular flux analyzer. LSD1 inhibition had no effect on oxidative phosphorylation (measured as oxygen consumption rate (OCR)). However, glycolysis, measured as proton efflux rate (PER), was significantly reduced from 102.88 ± 11.03 pmol/min to 54.17 ± 6.18 pmol/min (P &amp;lt;0.01) after 96 h of treatment with 1μM bomedemstat. With the combined treatments, we observed a reduction in both OCR and PER. This synergistic effect was further investigated by studying metabolite abundance using NMR spectrometry where co-treatment showed a reduction in metabolite abundance across a broad spectrum of metabolic pathways (Fig. 1). To determine if the observed combined effect of bomedemstat and venetoclax could enhance activity against AML cells in vivo, we employed a PDX model with two primary AML samples. In the first model system (monosomy 7), co-treatment resulted in a significant reduction in human CD45-positive cells as compared to vehicle (P &amp;lt;0.001) and single-agent treatments with venetoclax (P= 0.011) and bomedemstat (P &amp;lt;0.001). Similarly, in the second PDX model ( NPM1-mutated, FLT3-ITD positive), the co-treatment exhibited a significant reduction in leukemic burden in bone marrow as compared to vehicle (P=0.003) and single-agent treatments with venetoclax (P= 0.024) and bomedemstat (P &amp;lt; 0.001) (Fig. 1). Conclusion: The combination of bomedemstat and venetoclax had synergistic cytocidal effects on AML cell line and primary AML cells in vitro. It significantly reduced the leukemic burden in PDX AML models and synergistically downregulated cellular energy metabolism. These findings suggest that combining venetoclax with LSD1 inhibition holds promise as a combination treatment in AML and warrants further clinical investigation.

Real World Use of Azacitidine and Venetoclax in Acute Myeloid Leukemia in Frontline and Relapse/Refractory Settings: A Multicentric Study from French Auraml Group

Azacytidine and venetoclax combination regimen (AZA/VEN) is the standard of care in frontline acute myeloid leukemia (AML) settings for unfit to intensive chemotherapy patients. However, AZA/VEN is also associated with an increased hematological toxicity compared to azacytidine alone. In this context, alternative AZA/VEN regimens emerged progressively based on each physician experience and local procedures. Moreover, AZA/VEN is also recognized as a valuable therapeutic option in relapse/refractory settings. In this multicentric study, we aimed to evaluate the efficacy and safety of various AZA/VEN regimen in frontline and relapse/refractory (R/R) patients diagnosed with AML in real life setting. We retrospectively analyzed 331 patients from 11 different French centers (Saint-Etienne, Clermont-Ferrand, Lyon (Hopital Lyon Sud, Centre Léon Bérard), Vichy, Annecy, Chambery, Valence, Bourgoin-Jallieu, Grenoble, Roanne) in Auvergne Rhône Alpes (AURA) region, between January 2019 and February 2023. Composite complete remission was defined as in VIALE-A trial. Measurable residual disease (MRD) negativity was defined as ≤ 10 -3 by flow cytometry (on bone marrow) and/or ≤ 10 -4 for NPM1 by RT-qPCR. Overall, the entire cohort was composed of 186 and 146 patients in frontline and R/R settings respectively. In frontline setting, median age was 74.5 (19.1-86.1) and ELN 2022 risk groups were favorable, intermediate and unfavorable in 14.5, 16.7 and 68.8% respectively. De novo AML, MRC-AML, post-MPN/MDS AML and t-AML were 32.4%, 29.7%, 23.2% and 14.5% respectively. After 6 cycles, median CRc, morphologic leukemia free state (MLFS) and refractory rate were 66.4%, 5.4% and 28.2% respectively. Regarding probability of CRc, there was no significant difference between favorable and intermediate ELN 2022 risk group. Within unfavorable risk group, only complex karyotype was associated with lower CRc rate ( figure 1). Venetoclax dosage (400mg vs 100mg/d) and duration per cycle (28 days vs 21 days vs others) did not influence significantly CRc rate probability and OS. With a median follow-up of 7.3 months, median overall survival (OS) was 12.4 months (IC95%: 3.4-18.5). There was no significant difference in terms of OS according to ELN 2022 risk group classification ( figure 2). Among unfavorable risk group, only patients with unfavorable karyotype had a worst OS (median OS: 4.9 months), while patients with secondary AML mutations had similar OS than favorable/intermediate risk group (19.5 vs 20.2 vs 18.2 months, p=0.32). Reaching MRD negativity at any time during the first 6 cycles was associated with a better OS (26.1 vs 10.3 months, p&amp;lt;0.001). In responding patients, 22/122 withdrew VEN after a median of 5 cycles (range:1-17), while continuing AZA, mainly due to excessive hematological toxicity. Relapse free survival was similar between patients who stopped compared to those who continued up to progression. In multivariate analysis, only ECOG (continuous, HR= 1.52, p=0.04), IDH status (WT vs MUT: HR = 3.13, p=0.04, normal karyotype (yes vs no, HR=0.25, p=0.04) and CR type (CRMRD- vs other, HR= 0.21, p=0.001) significantly influence OS, whereas ELN 2022 did not In R/R settings, CRc rate was 43.8% and OS was 7.9 months. As in frontline setting, venetoclax dosage and duration per cycle did not influence CRs rate and OS. Febrile neutropenia (FN) occurred in 52.9%, 35.1% and 27.1% in cycle 1,2 and 3, respectively. Toxic death rates (TDR) range from 16.6% prior 2020 to 8.6% in 2021 and 6.25% from 2022 to 2023 (p=0.01). In or outpatient management for cycle 1 did not influence FN incidence. Azole prophylaxis did not significantly influence FN incidence. During cycle 1, shorter venetoclax duration regimens (&amp;lt;21 days/cycle) was associated with lower FN (41.5 vs 54.8%, p=0.042) compared to ≥21 days VEN cycle. In this real life settings study, our results suggest that outcome in frontline AZA/VEN treated patients is mainly driven by deep responses. Alternative AZA/VEN regimens were not associated with lower response rate and overall survival, while reducing venetoclax exposure during cycle 1 may reduce FN incidence without significant consequences on CR rate. TDR reduction through time suggest a learning curve and might be related to physician's skills improvement regarding AZA/VEN dosage schedules and supportive care management. An update with additional patients and longer follow-up will be presented at the meeting.

Early Results of the Phase I/II Study Investigating the All-Oral Combination of the Menin Inhibitor Revumenib (SNDX-5613) with Decitabine/Cedazuridine (ASTX727) and Venetoclax in Acute Myeloid Leukemia (SAVE)

Background: The interaction of menin with lysine methyltransferase 2A (KMT2A) is a dependency in acute leukemia caused by either rearrangement of the KMT2A (KMT2Ar) or Nucleoporin 98 ( NUP98r) genes, or mutation of the Nucleophosmin 1 gene ( NPM1mt). The menin inhibitor revumenib (previously SNDX-5613), is a potent, oral, selective inhibitor of the menin-KMT2A interaction with demonstrated safety and clinical activity in highly refractory acute leukemias (Issa GC, Nature 2023). KMT2Ar or NPM1mt leukemias are highly susceptible to induction of apoptosis through BCL2 inhibition, and dual Bcl-2 and menin inhibition led to synergistic activity in KMT2Ar or NPM1mt leukemia models (Carter BZ, Blood 2021). Therefore, we designed a phase I/II, investigator-initiated trial of the all-oral combination of revumenib, venetoclax and the hypomethylating agent ASTX727 in children and adults with relapsed/refractory (R/R) acute myeloid leukemia (AML) (NCT05360160). Methods: Patients (pts) with R/R AML or myeloid mixed-lineage acute leukemia (MPAL) aged 12 years and older were eligible.Dose escalation followed a 3+3 design. ASTX727 (decitabine/ cedazuridine) was administered at 35 mg/100 mg PO daily days 1-5, venetoclax at 400 mg (target dose) PO daily days 1-14, and revumenib 113 mg PO Q12h (dose level [DL] 0) or 163 mg PO Q12h (DL 1, used in phase II monotherapy), days 1-28 with either posaconazole or voriconazole (strong CYP3A4 inhibitors, for antifungal prophylaxis). Maintenance with revumenib monotherapy is planned following hematopoietic stem cell transplant (HSCT) for 1 year. For the first pt cohort, bone marrow examination was performed on cycle 1 day 14 and at the end of the cycle to assess for early morphologic remission and to inform future dose adjustments. Results: As of 7/20/2023, 8 pts were enrolled, 6 at DL0, and 2 at DL1. The median age was 27 years (range, 12-62 years), including 2 children (ages 12 and 16), 5 with KMT2Ar, 2 with NUP98r and 1 with NPM1mt. One pt had MPAL, and one had bone marrow and extramedullary disease. The median prior lines of therapy was 2.5 (range 1-4), 5 pts (63%) had prior venetoclax, 5 pts (63%) had a prior hypomethylating agent, 5 pts (63%) had prior HSCT, and one had prior menin inhibitor. The most common all-grade treatment-related adverse events (TRAEs) in ≥25% of pts were febrile neutropenia (63%), hyperphosphatemia (63%), nausea (63%), and AST/ALT elevation (25%). Grade ≥ 3 TRAEs were febrile neutropenia (63%), decreased platelets count (25%), and decreased neutrophil count (25%). There was 1 dose-limiting toxicity (DLT), grade 4 prolonged thrombocytopenia and neutropenia, at DL0 (resolved after dose hold), prompting enrollment of 3 additional pts, and escalation to DL1 when no additional DLTs were noted (1/6 pts with DLT). Pts at DL1 (N=2) have not completed the DLT period. There were no deaths due to TRAEs. No grade 3 or higher QTc prolongation occurred. Two pts had grade 2 differentiation syndrome (bone pain only), resolved with steroids. One pt ( NUP98r) had asymptomatic leukocytosis at the end of cycle 1 with neutrophilia, monocytosis, decrease in peripheral blasts, decrease in bone marrow blasts from 66% to 8%, and persistence of NUP98r by fluorescence in situ hybridization at 90% indicating differentiation. Seven of 8 pts are response-evaluable, and all 7 attained a morphologic remission (overall response rate of 100%) (Figure 1). Best responses achieved included a complete remission (CR) in 1 pt (including resolution of extramedullary disease), CR with partial hematologic recovery (CRh) in 1 pt, CR with incomplete platelet count recovery (CRp) in 3 pts, partial response (PR) in 1 pt, and 1 pt with morphologic leukemia free state (MLFS). Measurable residual disease was undetectable by flow (sensitivity at 10 -4) in 3 of 7 pts (43%). Three pts transitioned to HSCT following response, 2 are in continued remission, 1 has started maintenance and 1 pt died of transplant complications prior to starting maintenance. Enrollment is ongoing and updated data will be presented. Conclusions: Early results indicate acceptable safety and high efficacy of this combination in R/R myeloid leukemias with either KMT2Ar or NPM1mt or NUP98r. This study is ongoing with plans to establish the recommended phase 2 dose and optimize delivery of this combination.

Effectiveness of Intensive Chemotherapy with 7+3 Versus Vidaza and Venetoclax in Acute Myeloid Leukemia with Sequential Stem Cell Transplant: Results from Real-World Cohorts

Introduction: Intensive chemotherapy(IC) with cytarabine and anthracycline (7&amp;3) has been the backbone therapy for medically fit patients for the last 50 years. The use of Venetoclax and hypomethylating agent combination has be used in older and unfit patient populations. The Venetoclax combination regimen has improved outcomes in the selected group but has been characterized to be inferior to 7&amp;3 regimen among some studies. The role of stem cell transplant has not been previously discussed who receive IC versus Venetoclax based chemotehrapy Methods: Retrospective data was extracted using the TriNetX database. The primary endpoint was overall survival, and stem cell transplant in the two cohorts. Results: 5590 patients were identified in cohort-1 (7+3 cohort) and 2944 patients were identified in cohort-2 (Vidaza with Venetoclax cohort). Table-1 shows patient characteristics at baseline. Propensity-score-matching was used to balance the two cohorts. Patient count in each group after matching was 1393 (Table-2). Overall survival was 43.78% in cohort-1 versus 16.56 % in cohort-2, with HR of 0.510 (95% CI 0.457, 0.569) in favor of cohort 1 (Figure-1) at 5-year mark. Median OS for cohort-1 was 1055 days versus 289 days for cohort-2. Additional analysis was performed to determine number of patients who underwent a Stem Cell Transplant in each cohort and the effect of transplant on outcomes. 462 patients in cohort-1 and 358 patients in cohort-2 underwent transplant respectively. Overall survival was 51.29% in cohort-1 versus 61.64% in cohort-2, with HR of 1.180 (95% CI 1.027, 1.254) in favor of cohort-2 at 5-year mark. Median overall survival was not reached for either cohort at the data cut-off. Conclusions: Our retrospective analysis of a large patient cohort showed improved OS and decreased risk of death in patient's undergoing 7&amp;3 induction therapy as compared to Vidaza with Venetoclax. In patients who underwent stem cell transplant, the overall survival was statistically better in Vidaza with Venetoclax cohort as compared to 7&amp;3 induction therapy cohort. To the best of our knowledge, this is the first large database based study evaluating these two induction regimens accounting for stem cell transplantation.

Tumor Lysis Syndrome in Patients with AML Initiated on Venetoclax-Based Treatment without a Dose Ramp-up

Introduction Treatment with the combination of a hypomethylating agent (HMA) and venetoclax (VEN) is the standard of care for older patients (pts) with acute myeloid leukemia (AML). Venetoclax, an oral BCL-2 inhibitor, was initially approved for the treatment of chronic lymphocytic leukemia (CLL). In the early, pivotal studies assessing VEN safety, tumor lysis syndrome (TLS) was found to be a dose-limiting, potentially fatal toxicity. Dosing modifications were made to allow for safer initiation of the drug and, ultimately, a dose ramp-up was included in the dosing recommendation for CLL. Given the risk for TLS with VEN initiation for CLL, it was hypothesized that the risk would be the same, or potentially greater, when treating pts with AML. Therefore, studies of VEN in AML have included a dose ramp-up. Although reported rates of tumor lysis syndrome in these studies have been much lower than expected, a dose ramp-up for pts with AML is included in the package insert for VEN and continues to be utilized in clinical trials. Methods We conducted a retrospective analysis of pts ≥18 years of age with newly diagnosed or relapsed/refractory AML treated with a standard dose hypomethylating agent (HMA), either azacitidine or decitabine, and VEN without a dose ramp-up (initiated at 400mg daily or 100mg daily if on concomitant voriconazole or posaconazole) at Thomas Jefferson University Hospital from January 1, 2018- August 1, 2021. The primary objective of this study was to determine the rate of laboratory and clinical TLS within 72 hours after chemotherapy initiation, as defined by the Cairo-Bishop criteria (Table 1). The secondary objectives were (1) to identify clinical and laboratory risk factors for the development of TLS in pts with AML who were treated with HMA-VEN without a VEN dose ramp-up and (2) to compare rates of TLS in pts with AML who were treated with HMA-VEN without a VEN dose ramp-up to historical rates of TLS in pts who underwent a VEN dose ramp-up. In the Phase 3 study of azacitidine and venetoclax the rate of TLS during the VEN dose ramp-up period (days 1-3 of treatment) was 1% (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.). Results Ninety pts were included in the analysis. Seventy-one pts had newly diagnosed AML and 19 had relapsed/refractory disease. The majority of pts (58/90, 64%) had adverse risk disease by 2017 European LeukemiaNet Criteria. Median baseline white blood cell count at the time of therapy initiation was 3.25 (range 0.5-161.4). Sixty-one pts (68%) were receiving allopurinol prophylaxis at the time of HMA-VEN administration. Eighteen pts (20%) had a history of underlying renal disease. Fifteen pts (17%) were on medications, such as thiazide diuretics, aspirin, or levodopa, that can increase the risk of TLS at the time of treatment. Zero pts met the defined criteria for laboratory or clinical TLS. One pt developed acute kidney injury and one pt developed a cardiac arrhythmia (atrial fibrillation) but neither had concomitant evidence of laboratory TLS. Discussion In this retrospective study of 90 pts with newly diagnosed or relapsed/refractory AML who were treated with HMA-VEN without a VEN dose ramp-up no pts developed clinical or laboratory tumor lysis syndrome. This is comparable to the rate of TLS (1%) seen in the registration trial of HMA-VEN where pts were treated with VEN 100 mg on day 1, 200 mg on day 2, and 400 mg on day 3 and thereafter. Starting pts with AML on full dose VEN on day 1, regardless of white blood cell count, renal function, or concomitant medications, appears safe and could be implemented in clinical practice and clinical trials moving forward. This would simplify dosing for pts, pharmacists, and providers and allow for easier initiation of therapy.

Effect and Mechanism of NL-101 Combined with Bcl-2 Inhibitor Venetoclax in Acute Myeloid Leukemia

Background Acute myeloid leukemia (AML) is a kind of malignant clonal disease originating from hematopoietic stem cells with strong heterogeneity,with the highly recurrence rate and mortality rate.Although the application of hematopoietic stem cell transplantation and new targeted drugs has improved the prognosis of some patients in recent years, chemotherapy is still the most important treatment for AML. 50-80% of AML patients eventually relapsed after CR, with a low remission rate after relapse.There are reports that BCL-2 overexpression is a clinical feature of human hematologic malignancies that exacerbates the malignant state and drives apoptosis resistance phenotypes.Therefore, how to reduce treatment-related toxic and side effects and overcome tumor cell drug resistance is a hot and difficult topic in current AML treatment. Venetoclax (ABT-199) is an oral, selective Bcl-2 inhibitor. It showed strong anti-leukemia activity in preclinical stage, but its clinical efficacy as a single drug for AML was limited.Nl-101 (also known as EDE-S101)is a new small molecule compound formed by covalently linking the DNA damage core group of Bendamustine hydrochloride with the inhibition core group of histone deacetylase of the histone inhibitor Volinostat (also known as SAHA).Our previous study found that NL-101 single drug can reduce tumor load and prolong survival of AML mice in vivo.In this study, we used Venetoclax in combination with NL-101 to understand its efficacy and safety in vitro and in vivo, and to explore the synergies and mechanisms of action between the two drugs, providing new effective options for the treatment of AML patients. Purpose To explore the efficacy of Bcl-2 inhibitor Venetoclax (ABT-199) and a novel small molecule compound NL-101 in the treatment of acute myeloid leukemia（AML） and the related mechanisms. Experimental Design AML cell lines and primary patient samples were collected and used for cell proliferation assay. MTS colorimetric assay was used to determine the effect of Venetoclax alone or in combination with NL-101 on the growth of AML cells，the combination index (CI) was calculated to evaluate the synergistic effect of the two drugs. Cell death induced by Venetoclax alone or in combination with NL-101 was analyzed by Annexin V-FITC/ Propyl -iodide staining and flow cytometry. Western blot was used to analyze the protein expression levels of MAPK/ERK, PI3K/AK/GSK pathway members and BCL-2 family members.The MV4-11-derived xenograft mouse model was used to assess in vivo efficacy of the combination of venetoclax and NL-101. Results Venetoclax alone and in combination with NL-101 can inhibit the growth of AML cell lines and primary cells to a certain extent, and the combination of drugs has a synergistic effect. Venetoclax combined with NL-101 effectively synergistically induced apoptosis in AML cell lines and primary patient samples. Venetoclax combined with NL-101 can down-regulate the expression of pro-survival proteins in the Bcl-2 family, such as McL-1, Bcl-2, and Bcl-XL, and up-regulate the expression of pro-apoptotic proteins, such as BIM, BID, and BAX/BAK. Phosphorylated ERK, AKT, and JNK expressions were eliminated by venetoclax in combination with NL-101. In vivo results suggest that Venetoclax combined with NL-101 has the potential to treat AML. Conclusions Venetoclax combined with NL-101 can synergistically inhibit the growth and promote apoptosis of AML cell lines and primary AML cells. In vivo experiments demonstrated that Venetoclax combined with NL-101 can synergistically reduce tumor load and prolong survival time in AML mice. These results suggest that Venetoclax in combination with NL-101 is effective in the treatment of acute myeloid leukemia.

Inpatient Versus Outpatient Administration of Hypomethylating Agents and Venetoclax in Acute Myeloid Leukemia

Background: Venetoclax (VEN) in combination with hypomethylating agents (HMAs) is the standard of care for newly diagnosed AML patients who are older and determined to be unfit for intensive induction chemotherapy. The decreased intensity of HMA/VEN affords the possibility of outpatient management provided appropriate monitoring can be done for tumor lysis syndrome, persistent cytopenias, and infection risk inherent to initial AML directed therapy. We evaluated the incidence of TLS, infection risk, and transfusion needs in patients with AML treated with HMA/VEN at our institution who initiated therapy as an inpatient versus outpatient to determine the safety and feasibility of initial outpatient HMA/VEN therapy. Methods: This was a retrospective Lifespan IRB approved review of patients diagnosed with AML from 12/1/2016 to 12/1/2021. Patients who received HMA/VEN for AML, either as frontline or as relapsed/refractory therapy, were included. Patients' records were reviewed for demographic, leukemia specific, and patient specific outcomes. TLS was based on Cairo Bishop classification. Fisher exact/Pearson chi-squared and Mann Whitney U tests were used to determine statistical differences for categorical and continuous variables, respectively, while the log rank test was used for overall survival (OS). Results: Between 12/1/2016 and 12/1/2021, 122 patients were initiated on HMA/VEN with 73 patients treated frontline (24 inpatient, 49 outpatient) and 49 treated in the relapsed/refractory setting (11 inpatient, 38 outpatient). In the frontline setting, there was no difference in demographic data between those patients who initiated HMA/VEN inpatient versus outpatient while relapsed/refractory patients who were initiated inpatient were more likely to have a higher Charlson Comorbidity Index than that of those outpatients ( Table 1). In terms of leukemia specific factors, frontline patients who received inpatient HMA/VEN were more likely to have a higher WBC at diagnosis. No differences in leukemia specific factors were seen amongst relapsed/refractory patients ( Table 1). No difference in infections, TLS risk, or transfusion needs was seen amongst frontline patients who received HMA/VEN inpatient vs outpatient. Relapsed/refractory patients who received HMA/VEN inpatient had an increased risk of febrile neutropenia and required more pRBC and platelet transfusions than patients who received HMA/VEN outpatient. ( Table 1). OS did not differ for frontline use of HMA/VEN amongst patients who initiated treatment inpatient versus outpatient [11 mo, (1-50) versus 6 mo (0-26), p 0.64]. In relapsed/refractory patients, those who were initiated on HMA/VEN outpatient had significantly higher OS (12.5 mo, range 0-76 mo) than those who were initiated on HMA/VEN inpatient (6 mo, range 2-50 mo, p 0.02) likely secondary to the increase in consolidative allo-SCT amongst outpatient relapsed/refractory HMA/VEN patients versus inpatient relapsed/refractory HMA/VEN patient (19 patients vs 1 patient, p 0.02). Conclusion: Patients at our institution were able to be initiated on HMA/VEN as an outpatient for AML treatment without any perceived increase in TLS or infection risk in both the frontline and relapsed/refractory settings. Center and patient specific factors, such as travel distance and ability to administer supportive care, should be considered with outpatient administration of HMA/VEN.