High Levels Of Bcl-2 Research Articles

Differentiation State Plasticity As a Mechanism of BCL2 Inhibitor Resistance in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is composed of a heterogeneous blast population that exhibits characteristics of normal myeloid cells, including immature, HSC-like cells and differentiated, monocyte-like cells. The balance of immature and differentiated blasts within a patient is important clinically, as immature AML blasts are acutely sensitive to treatment with venetoclax, a BH3 mimetic that blocks the anti-apoptotic activity of BCL2. BCL2-inhibitor-based therapeutic strategies are the standard of care among elderly patients with untreated or relapsed/refractory AML. While these strategies result in a remission rate of nearly 70%, the majority of patients who do achieve remission ultimately relapse. Frequently, the relapsed disease is composed of differentiated, monocyte-like blasts. Given the importance of BCL2 inhibitors to the clinical management of AML, it is critical to understand the mechanisms by which BCL2 inhibitors result in monocytic relapse. Disease relapse has been traditionally thought to emerge from intrinsically resistant cells that confer a survival advantage under therapeutic pressure. Several studies have suggested that differentiated AML blasts have a survival advantage in the presence of BCL2 inhibition due to a reliance on anti-apoptotic proteins other than BCL2. Immature AML blasts express high levels of BCL2, whereas blasts that are primed for lympho-myeloid or granulocytic-monocytic progenitor cell fates express high levels of MCL-1 and BCL-XL, respectively. However, these studies do not explain why patients with dominant, differentiated subclones frequently achieve long-lasting remission with BCL2-inhibitor-based strategies. Therefore, simple selection of differentiated blasts appears to be an insufficient model to describe BCL2 inhibitor resistance. Alternative models of therapeutic resistance have been identified where cancer cells adapt to a resistant phenotype and acquire a survival advantage under therapeutic pressure. In the context of BCL2 inhibitor resistance, it is unclear the extent to which the emergence of differentiated blasts is the result of selective pressure or blast differentiation state plasticity in response to BCL2 inhibition. In our work, we have investigated the adaptive mechanism of BCL2 inhibition resistance in AML. We have explored this mechanism using an AML cell line model in which immature AML cells continuously generate differentiated AML cells (OCI-AML8227). We used fluorescence-activated cell sorting to isolate immature CD34+/CD38- OCI-AML8227 cells. After 72 hours in liquid culture, we observed that these cells recapitulated a pool of differentiated CD34-/CD38+/CD14+ cells. Furthermore, we used single cell ATAC sequencing to confirm that the unfractionated pool of OCI-AML8227 cells display chromatin accessibility profiles resembling cells across the continuum of myeloid differentiation. To understand whether this model is appropriate to investigate the differentiation-state-specific differences in sensitivity to BLC2 inhibition, we measured the viability of immature CD34+ and differentiated CD34- OCI-AML8227 cells following BCL2 inhibition. Consistent with previous studies, immature CD34+ cells were significantly more sensitive to BCL2 inhibition than differentiated CD34- cells. In addition, BLC2 inhibition of unfractionated OCI-AML8227 cells resulted in an 82% increase in the number of differentiated CD34-/CD38+ cells. While the number of immature CD34+/CD38- cells was reduced following BCL2 inhibition, it was only reduced by 27%. Furthermore, there was no change in the apoptosis marker Annexin V among immature CD34+/CD38- cells. These results support an adaptive resistance model where AML cells acquire resistance through differentiation state plasticity. We will explore this mechanism by following the temporal dynamics of immature and differentiated blasts following BCL2 inhibition. Using single cell barcoding, we will not only be able to trace the trajectories of these populations but also investigate transcriptional adaptation of the immature and differentiated populations. With an understanding of the adaptive mechanism of resistance to BCL2 inhibition, we can nominate alternative targets to prevent the outgrowth of resistance populations and design more effective, rational BLC2-inhibitor-based therapeutic strategies.

Venetoclax Combined with Hag Regimen in Newly Diagnosed ETP-ALL: Interim Analysis of a Prospective, Multicenter, Phase 2 Trial

Background: Early T-cell precursor (ETP) acute lymphoblastic leukemia (ALL) is identified as a subset of T-lineage ALL with a unique immunophenotype and genome profile in 2009. Patients with ETP-ALL have a relatively poor outcome with standard chemotherapy and studies showed that therapies utilized for myeloid neoplasms may be effective in ETP-ALL given the relatively high frequency of molecular myeloid features. In recent years, targeting of the antiapoptotic protein BCL-2 has emerged as an effective approach in acute myeloid leukemia (AML). Preclinical studies reported that ETP-ALL cells also express high level of BCL-2 and are sensitive to the BCL-2 inhibitor venetoclax (VEN). However, the clinical efficacy of venetoclax combination therapies in ETP-ALL patients is still limited. Our data showed that homoharringtonine (HHT) showed strong synergistic effect with VEN in ETP-ALL (2022 ASH abstract #170604). We therefore conducted a study of combination of VEN and HHT-based HAG regimen (V-HAG regimen) in newly diagnosed ETP-ALL patients. The aim of this study was to investigate the activity and tolerability of V-HAG regimen for newly diagnosed ETP-ALL. Methods: In this prospective, multicenter, phase 2 clinical trial conducted in 7 different hematological centers in China, eligible patients were newly diagnosed ETP-ALL(aged≥16 years old) based on the 2016 WHO classification with an Eastern Cooperative Oncology Group performance status of 0-2. Patients received venetoclax (100mg on day1, 200mg on day 2 and 400mg on day 3-14, if the blast cells in bone marrow was more than 5% on day 14, the patient continued to receive venetoclax 400mg until day 28), homoharringtonine (1.4 mg/m 2,2mg maximum daily, intravenously daily from on d1-10), Low-dose cytarabine (10 mg/m2 subcutaneously every 12h on d1-14), and G-CSF (100ug/m 2 daily on d1-14). Venetoclax dose was reduced by at least 50% in the patients receiving concomitant moderate or strong CYP3A4 inhibitors (such as azole antifungals et al). The primary endpoint was the rate of composite complete remission (CRc) after one cycle of induction treatment, including CR and CR with incomplete count recovery (CRi). Secondary endpoints included bone marrow (BM) minimal residual disease (MRD) by flow cytometry after one cycle of induction treatment, overall survival (OS), event-free survival (EFS), and adverse events (AEs). Here, we report results of the interim analysis. Results: Between July 2022 and July 2023, we enrolled 18 patients with newly diagnosed ETP-ALL in 7 different hematological centers. 14 of 18 patients (77.8%) were male and 22.2% were female, with a median age of 51 years (range, 29 to 73 years). As of July 31, 2023, 15 patients were available for the assessment of treatment results. Among the 15 patients, 14 patients were given standard V-HAG regimen as showed above and one 73-year-old patient, who had interstitial pneumonia at the onset of ETP-ALL, was given dose-reduced V-HAG regimen. To our surprise, after the first cycle of treatment with V-HAG, all the 15 patients achieved CRc (100% CRc rate), including the 73-year-old patient with dose-reduced regimen. Then, after one to three cycles of consolidation treatment with V-HAG regimen, 7 of 15 patients have proceeded to allo-HSCT so far. With a median follow-up of 7.7 months, both the median OS and EFS were not achieved. In fact, to date, except for the 73-year-old patient who eventually relapsed (6% blasts in BM after receiving a second cycle of dose-reduced V-HAG regimen as consolidation) and died of respiratory failure, all the other 14 patients are remaining alive and event-free. For the adverse events, the most common toxicities were grade 4 neutropenia (93.33%), grade 4 thrombocytopenia (73.33%) and grade 3 febrile neutropenia (26.67%). No early death occurred. Conclusions: Venetoclax Combined with HAG regimen achieved an 100% CRc rate in newly diagnosed ETP-ALL to date. Enrollment is ongoing to assess its efficacy and safety in treating newly diagnosed ETP-ALL. This therapy is a promising and well-tolerated regimen in newly diagnosed ETP-ALL patients. Trial registration: The trial was registered in the Chinese Clinical Trial Register with the registration number ChiCTR2200061708.

High-Glucose-Induced Injury to Proximal Tubules of the Renal System Is Alleviated by Netrin-1 Suppression of Akt/mTOR.

The kidneys have a high level of Netrin-1 expression, which protects against some acute and chronic kidney disorders. However, it is yet unknown how Netrin-1 affects renal proximal tubule cells in diabetic nephropathy (DN) under pathological circumstances. Research has shown that autophagy protects the kidneys in animal models of renal disease. In this study, we looked at the probable autophagy regulation mechanism of Netrin-1 and its function in the pathogenesis of DN. We proved that in HK-2 cell, high blood sugar levels caused Netrin-1 to be downregulated, which then triggered the Akt/mTOR signaling pathway and enhanced cell death and actin cytoskeleton disruption. By adding Netrin-1 or an autophagy activator in vitro, these pathogenic alterations were reverted. Our results indicate that Netrin-1 stimulates autophagy by blocking the Akt/mTOR signaling pathway, which underlies high-glucose-induced malfunction of the renal proximal tubules. After HK-2 cells were incubated with Netrin-1 recombination protein and rapamycin under HG conditions for 24 h, the apoptosis was significantly reduced, as shown by the higher levels of Bcl-2, as well as lower levels of Bax and cleaved caspase-3 (P = 0.012, Cohen's d = 0.489, Glass's delta = 0.23, Hedges' g = 0.641). This study reveals that targeting Netrin-1-related signaling has therapeutic potential for DN and advances our knowledge of the processes operating in renal proximal tubules in DN.

Low-Dose Venetoclax-Dexamethasone in t(11;14) Positive Relapsed and Refractory Multiple Myeloma; Interim Results from the Ongoing, Danish, Investigator-Initiated, Open Label, Phase 2 Victoria Study

Background Venetoclax (VEN) is an oral BCL-2 inhibitor that is effective for the treatment of multiple myeloma (MM) with t(11;14), expressing high levels of BCL-2. In a previous phase 1 study, VEN was given to 66 patients [30 with t(11;14)] with relapsed and refractory MM (RRMM) at 300, 600, 900 or 1200 mg in dose-escalation cohorts and 1200 mg in the expansion cohort, without reaching a maximum tolerated dose; the overall response rate (ORR) in t(11;14) positive patients was 40% (Kumar et al. 2017). Later, in a phase 1-2 study, 800 mg VEN with 40/20 mg weekly dexamethasone (DEX) was explored in 51 patients with RRMM and t(11;14) with an overall response rate (ORR) of 48-60% (Kaufman et al. 2021). Here, we report the interim results of an ongoing, Danish, investigator-initiated, open label, phase 2 study which is testing the safety and efficacy of low-dose VEN-DEX in RRMM. Methods Patients with RRMM, at least one prior line of therapy and t(11;14) were included in the study between 1 st July 2020 and data cutoff in 31 st July 2022. VEN was administered orally, once daily at a dose of 400 mg. DEX was administered orally, once weekly at a dose of 20 mg. Treatment continued until progressive disease or unacceptable toxicity. After discontinuation of treatment, patients were followed for 24 months. The primary endpoint of the study was ORR, key secondary endpoints were progression-free survival (PFS) and overall survival (OS). Grade three or higher adverse events (AE) were recorded. Results Patient characteristics At the time of data cutoff in July 2023, twenty-six patients were included in the study. The median (IQR) age was 74 (63-82) years. The median time from diagnosis was 4 (1-7) years. The median (IQR) number of prior lines of therapy was 3 (2-5). 46% of patients were males. At screening, 56% had anemia, 7% had renal failure, 15% had hypercalcemia according to the CRAB criteria. Performance status was 0 in 40% and 1-2 in 60% of patients. 47% of patients had ISS I, 19% had ISS II, 33% had ISS III. Besides t(11;14), present in all patients, 7% of patients had high-risk cytogenetic abnormalities, defined as the presence of t(4;14), t(14;16) or del(17p). 55% of patients had been treated with high dose melphalan and autologous stem cell transplantation, 92% had been exposed to an IMiD, 96% to a proteasome inhibitor, 70% to daratumumab. 29% were triple class-refractory, 15% were penta-drug refractory. Safety One patient had a grade 4 ventricular ulcer, besides this there were no grade 4 or 5 AEs. The most frequent grade 3 AE was infection, detected in 9 (35%) patients. Of these, there were 3 cases of pneumonia, 3 cases of COVID-19, 1 case of influenza, 1 case of gastroenteritis, and 1 case of fever of unknown focus. Other grade 3 AEs related to venetoclax were dehydration in 2 patients, thrombocytopenia in 1 patient, and nausea in 1 patient. The dose of venetoclax was reduced in 3 patients, to 200 mg daily in each case. Efficacy Ten of 25 evaluable patients had partial response or better, resulting in an ORR of 40% (Figure 1A). Four percent of patients achieved stringent complete response, 16% very good partial response and 20% partial response. In subgroup analysis, worse responses were observed in patients with creatinine >177 µmol/L (n=2), high-risk cytogenetics (n=2), triple-class refractory (n=7) and penta-drug refractory (n=3) disease. In responding patients, the median duration of response (DOR) was not reached. The estimated percentage of patients still responding to therapy was 74% after one year and 55% after two years. The median (95% CI) PFS in the intention-to treat population was 5.5 (3.7; not available) months (Figure 1B). Median OS was not reached. The estimated percentage of patients still alive was 90% after one year. Conclusion: Based on this interim analysis, low-dose VEN-DEX in patients with RRMM and t(11;14) has a convenient safety profile and an efficacy comparable to previously tested VEN and VEN-DEX regimens with higher VEN doses. Updated results will be presented at the meeting.

Walnut Kernel Oil and Defatted Extracts Enhance Mesenchymal Stem Cell Stemness and Delay Senescence.

Decreased stemness and increased cellular senescence impair the ability of mesenchymal stem cells (MSCs) to renew themselves, change into different cell types, and contribute to regenerative medicine. There is an urgent need to discover new compounds that can boost MSCs' stemness and delay senescence. Therefore, this study aimed to investigate the impact of walnut kernel oil (WKO) and defatted (WKD) extracts on bone marrow (BM)-MSC stemness and senescence. Premature senescence and inflammation were induced in BM-MSCs using H2O2 and LPS, respectively. Phytochemical constituents of WKO and WKD extracts were detected by HPLC. The stemness (proliferation and migration), senescence-related markers (p53, p21, SIRT1, and AMPK), oxidative stress/antioxidant markers, inflammatory cytokines, and cell cycle of BM-MSCs were measured by MTT assay, qPCR, ELISA, and flow cytometry. WKO and WKD extracts improved rat BM-MSC stemness, as evidenced by (1) increased cell viability, (2) decreased apoptosis (low levels of Bax and caspase3 and high levels of Bcl2), (3) upregulated MMP9 and downregulated TIMP1 expression, and (4) cell cycle arrest in the G0/G1 phase and declined cell number in the S and G2/M phases. Additionally, WKO and WKD extracts reduced rat BM-MSC senescence, as indicated by (1) decreased p53 and p21 expression, (2) upregulated expression and levels of SIRT1 and AMPK, (3) reduced levels of ROS and improved antioxidant activity (higher activity of CAT, SOD, and GPx and upregulated expression of NrF2 and HO-1), and (4) declined levels of TNFα, IL1β, and NF-κB. When compared to the WKO extract, the WKD extract had a greater impact on the induction of stemness and reduction of senescence of BM-MSCs due to its stronger antioxidant activity, which could be attributed to its higher levels of flavonoids and phenolic compounds, as detected by HPLC analysis. WKO and WKD extracts enhance rat BM-MSC stemness and protect them from senescence, suggesting their potential use as enhancers to increase MSCs' therapeutic efficacy.

Rosemarinic acid protects β-cell from STZ-induced cell damage via modulating NF-κβ pathway

Rosmarinic acid (RA), a natural ester phenolic compound, is known to have antioxidant and anti-inflammatory properties. RA has also been reported to exhibit a hypoglycemic effect; however, the mechanisms underlying this effect have yet to be investigated. Therefore, the present study focused on the anti-diabetic effects and mechanism of RA in INS-1 cells using in vitro model. Streptozotocin (STZ) at a concentration of 3 mM was applied to INS-1 cells for 4 h to create a diabetic model. The cells were pretreated for 24 h with various concentrations (1 and 2.5 μM) of RA. The Cell viability, glucose-stimulated insulin secretion (GSIS), glucose uptake, lipid peroxidation, reactive oxygen species (ROS), apoptosis, and protein expression of Bcl-2, NF-κB, 1L-1β, and PARP were assessed. Results showed that STZ-treated INS-1 cells exhibited reduced cell viability, insulin release, insulin content, glucose uptake, and elevated MDA and ROS levels. Cells pretreated with RA maintained the function and morphology of β-cells against STZ-induced damage. Moreover, RA sustained high protein expression levels of Bcl-2 and low expression levels of NF-κB, IL-1β, and PARP. In conclusion, RA preserved β-cells function against STZ-induced damage by altering NF-κB and Bcl-2 pathways.

Targeting Oncogenic Mutant p53 and BCL-2 for Small Cell Lung Cancer Treatment.

Through a unique genomics and drug screening platform with ~800 solid tumor cell lines, we have found a subset of SCLC cell lines are hypersensitive to venetoclax, an FDA-approved inhibitor of BCL-2. SCLC-A (ASCL1 positive) and SCLC-P (POU2F3 positive), which make up almost 80% of SCLC, frequently express high levels of BCL-2. We found that a subset of SCLC-A and SCLC-P showed high BCL-2 expression but were venetoclax-resistant. In addition, most of these SCLC cell lines have TP53 missense mutations, which make a single amino acid change. These mutants not only lose wild-type (WT) p53 tumor suppressor functions, but also acquire novel cancer-promoting activities (oncogenic, gain-of-function). A recent study with oncogenic mutant (Onc)-p53 knock-in mouse models of SCLC suggests gain-of-function activity can attenuate chemotherapeutic efficacy. Based on these observations, we hypothesize that Onc-p53 confers venetoclax resistance and that simultaneous inhibition of BCL-2 and Onc-p53 induces synergistic anticancer activity in a subset of SCLC-A and SCLC-P. We show here that (1) down-regulation of Onc-p53 increases the expression of a BH3-only pro-apoptotic BIM and sensitizes to venetoclax in SCLC-P cells; (2) targeting Onc-p53 by the HSP90 inhibitor, ganetespib, increases BIM expression and sensitizes to venetoclax in SCLC-P and SCLC-A cells. Although there are currently many combination studies for venetoclax proposed, the concept of simultaneous targeting of BCL-2 and Onc-p53 by the combination of venetoclax and HSP90 inhibitors would be a promising approach for SCLC treatment.

Targeting BCL2 Overcomes Resistance and Augments Response to Aurora Kinase B Inhibition by AZD2811 in Small Cell Lung Cancer.

Therapeutic resistance to frontline therapy develops rapidly in small cell lung cancer (SCLC). Treatment options are also limited by the lack of targetable driver mutations. Therefore, there is an unmet need for developing better therapeutic strategies and biomarkers of response. Aurora kinase B (AURKB) inhibition exploits an inherent genomic vulnerability in SCLC and is a promising therapeutic approach. Here, we identify biomarkers of response and develop rational combinations with AURKB inhibition to improve treatment efficacy. Selective AURKB inhibitor AZD2811 was profiled in a large panel of SCLC cell lines (n = 57) and patient-derived xenograft (PDX) models. Proteomic and transcriptomic profiles were analyzed to identify candidate biomarkers of response and resistance. Effects on polyploidy, DNA damage, and apoptosis were measured by flow cytometry and Western blotting. Rational drug combinations were validated in SCLC cell lines and PDX models. AZD2811 showed potent growth inhibitory activity in a subset of SCLC, often characterized by, but not limited to, high cMYC expression. Importantly, high BCL2 expression predicted resistance to AURKB inhibitor response in SCLC, independent of cMYC status. AZD2811-induced DNA damage and apoptosis were suppressed by high BCL2 levels, while combining AZD2811 with a BCL2 inhibitor significantly sensitized resistant models. In vivo, sustained tumor growth reduction and regression was achieved even with intermittent dosing of AZD2811 and venetoclax, an FDA-approved BCL2 inhibitor. BCL2 inhibition overcomes intrinsic resistance and enhances sensitivity to AURKB inhibition in SCLC preclinical models.

Protective effects of ferulic acid against ionizing radiation-induced oxidative damage in rat lens through activating Nrf2 signal pathway.

To examine the protection of ferulic acid (FA) against ionizing radiation (IR)-induced lens injury in rats, as well as the underlying mechanisms. FA (50 mg/kg) was administered to rats for 4 consecutive days before they were given 10 Gy γ-radiation, as well as for 3 consecutive days afterward. Two weeks after radiation, the eye tissues were collected. Histological alterations were evaluated by hematoxylin-eosin staining. Enzyme linked immunosorbent assay (ELISA) was utilized to assess the activities of glutathione reductase (GR) and superoxide dismutase (SOD), as well as the levels of glutathione (GSH) and malondialdehyde (MDA) in the lenses. The protein and mRNA levels of Bcl-2, caspase-3, Bax, heme oxygenase-1 (HO-1), and glutamate-cysteine ligase catalytic subunit (GCLC) were quantified using Western blot and quantitative reverse transcription polymerase chain reaction, respectively. With nuclear extracts, the nuclear factor erythroid-2 related factor (Nrf2) protein expressions in the nuclei were also measured. Rats exposed to IR showed lens histological alterations which could be alleviated by FA. FA treatment reversed apoptosis-related markers in IR-induced lens, as evidenced by lower levels of Bax and caspase-3 and higher level of Bcl-2. Furthermore, IR induced oxidative damage manifested by decreased GSH level, increased MDA level, and decreased SOD and GR activities. FA boosted nuclear translocation of Nrf2 and increased the expressions of HO-1 and GCLC to inhibit oxidative stress, as evidenced by an increase in GSH, a decrease in MDA, and an increase in GR and SOD activities. FA may work well in preventing and treating IR-induced cataract through promoting the Nrf2 signal pathway to attenuate oxidative damage and cell apoptosis.

Abstract A07: Single-cell proteomic assessment of FLT3-ITD AML landscape identifies distinct resistance patterns

Abstract Acute myeloid leukemia (AML) represents a heterogeneous hematopoietic disorder characterized by accumulation of immature hematopoietic precursors with differentiation block. FLT3 internal tandem duplications (FLT3-ITD) are commonly occurring genetic alterations in AML and are associated with poor prognosis. Preclinical studies showed combining FLT3 inhibitor with MDM2 inhibitor, which is a negative regulator of p53, was synergistic in FLT3-ITD/TP53 wild type (WT) AML. We performed single-cell proteomic evaluation of leukemia landscape in FLT3-ITD patients treated with FLT3i and MDM2i to assess proteomic profiles associated with response and resistance. We performed CyTOF analysis of leukemia cells in serially collected samples from six FLT3-ITD and TP53 WT AML patients following treatment with FLT3i+MDM2i treatment. Using 51 features assessed in CyTOF, we first performed UMAP dimension reduction and clustering to identify distinct cells in leukemia compartment. Notably, the frequencies of blasts identified through CyTOF data analysis were compatible with clinical lab reports. In line with previous reports, we also detected that NPM mutant AML cells did not express CD34. Of note, CD34+ and CD34- leukemia cells were clustered together in NPM1 mut patients, indicating that they have overlapping proteomic profiles. Interestingly, the CD34+ leukemia cells were eliminated at the early time points in CR patients while the CD34- leukemia cells were still detectable after two months. On the other hand, CD34+ leukemia cells in nonresponders(NR) persisted despite therapy. These findings indicate CD34+ leukemia cells were more sensitive to the treatment compared to NPM1 mutant CD34- leukemia cells. Next, we interrogated leukemia proteomic landscape in serial samples, evaluated the therapy-induced alterations in proteomic profiles and sought to identify potential adaptive mechanisms. To this end, we performed differential expression analysis and observed that signaling pathways (p-4EBP1, p-GSK3, p-MEK1/2, p-S6) and differentiation markers(CD33, CD68,CXCR4 HLADR) were more enriched on day 8 post treatment in NR patients, revealing that compensatory signaling activity, phenotypic profiles and differentiation status could be associated with therapy response. Moreover, we found that leukemia cells in NR patients had distinct phenotypic profiles(CD11b,CD68,CXCR4), higher levels of anti-apoptotic molecules (BCL2,MCL1) and enriched survival pathways (p-GSK3, YTHDF2) compared to baseline. In contrast, we did not observe rebound increases in CR patients post-therapy. These findings demonstrate high levels of BCL2 and MCL1 and preferential survival of more differentiated cells may be associated with therapy resistance and treatment failure in NR patient treated with MDM2i and FLT3i. In conclusion, multiplexed single-cell proteomic analysis permitted longitudinal monitoring of leukemia landscape and identified proteomic alterations associated with therapy resistance. Citation Format: Li Li, Muharrem Muftuoglu, Mahesh Basyal, Naval Daver, Michael Andreeff. Single-cell proteomic assessment of FLT3-ITD AML landscape identifies distinct resistance patterns [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A07.

Abstract 5497: Venetoclax and dinaciclib elicit synergisticpreclinicalefficacy against high-risk B-cell leukemia

Abstract Hypodiploid acute lymphoblastic leukemia (ALL) is an aggressive blood cancer with a poor prognosis despite intensive chemotherapy or stem cell transplant. Children and adolescents with positive end-of-induction minimal residual disease (MRD) have an overall survival lower than 30%. However, data regarding therapeutic alternatives for this disease is nearly non-existent, emphasizing the critical need for new or adjunctive therapies that can improve outcomes. We previously reported on the therapeutic efficacy of venetoclax (ABT-199) in hypodiploid B-ALL but with limitations as monotherapy (Diaz-Flores, Cancer Research, 2019). Accordingly, hypodiploid B-ALL patient derived xenografts treated with Venetoclax developed therapeutic resistance over time. To better understand the mechanism of resistance that arises following inhibition of BCL-2, we first generated a hypodiploid cell line (NALM-16) deficient in BCL-2. Given that BCL-2 plays an essential role in B-cell function standard CRISPR/Cas9 knockout strategies greatly impaired survival. We overcame this limitation by using a magnetic levitation system (the LeviCell by LevitasBio). Analysis of the surviving clones identified an increase in MCL1 (Fig. 1A). We then harnessed unbiased drug screen (>1800 bioactive compounds) followed by selection of synergistic drug combinations identified Dinaciclib as an efficient drug combination with Venetoclax in eliminating leukemic cells in vitro. Interestingly, mechanistic studies identified that Dinaciclib, through transcription regulation (RNApol II) significantly modulates MCL-1. Data using knock-outs and knock-ins of MCL-1 indicated how MCL-1 only becomes essential for survival when Venetoclax is present. Preclinical studies using patient derived xenografts indicated that, while none of the drug monotherapies achieved complete blast elimination, venetoclax + dinaciblib led to complete resolution of the leukemic burden within all solid organs analyzed (Fig. 1B). This combination therapy was relatively well tolerated. This study also indicates that high BCL-2 and MCL-1 levels with concomitant BIM, BAD and/or PUMA levels, represent biomarkers for response to this combination. In summary, our study identified a highly synergistic drug combination, venetoclax and dinaciclib, for the treatment of hypodiploid B ALL, an aggressive leukemia with few effective current therapies. Finally, the promising results presented in this study may prompt further studies to support the inclusion of hypodiploid and other B-ALL patients to clinical trials combining phase I/II drugs against BCL-2 (mainly venetoclax) and CDK9 (dinaciclib, alvocidib, flavopiridol, SNS-032) or MCL-1 (MIK-665). Citation Format: Ernesto Diaz-Flores, Holly Pariury, Mignon Loh. Venetoclax and dinaciclib elicit synergisticpreclinicalefficacy against high-risk B-cell leukemia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5497.

The anti-estrogen receptor drug, tamoxifen, is selectively Lethal to P-glycoprotein-expressing Multidrug resistant tumor cells

BackgroundP-glycoprotein (P-gp), a member of the ATP Binding Cassette B1 subfamily (ABCB1), confers resistance to clinically relevant anticancer drugs and targeted chemotherapeutics. However, paradoxically P-glycoprotein overexpressing drug resistant cells are “collaterally sensitive” to non-toxic drugs that stimulate its ATPase activity.MethodsCell viability assays were used to determine the effect of low concentrations of tamoxifen on the proliferation of multidrug resistant cells (CHORC5 and MDA-Doxo400), expressing P-gp, their parental cell lines (AuxB1 and MDA-MB-231) or P-gp-CRISPR knockout clones of AuxB1 and CHORC5 cells. Western blot analysis was used to estimate P-gp expression in different cell lines. Apoptosis of tamoxifen-induced cell death was estimated by flow cytometry using Annexin-V-FITC stained cells. Oxidative stress of tamoxifen treated cells was determined by measuring levels of reactive oxygen species and reduced thiols using cell-permeant 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) and 5,5-dithio-bis-(2-nitrobenzoic acid) DTNB, respectively.ResultsIn this report, we show that P-gp-expressing drug resistant cells (CHORC5 and MDA-Doxo400) are collaterally sensitive to the anti-estrogen tamoxifen or its metabolite (4-hydroxy-tamoxifen). Moreover, P-gp-knockout clones of CHORC5 cells display complete reversal of collateral sensitivity to tamoxifen. Drug resistant cells exposed to low concentrations of tamoxifen show significant rise in reactive oxygen species, drop of reduced cellular thiols and increased apoptosis. Consistent with the latter, CHORC5 cells expressing high levels of human Bcl-2 (CHORC5-Bcl-2) show significant resistance to tamoxifen. In addition, the presence of the antioxidant N-acetylcysteine or P-gp ATPase inhibitor, PSC-833, reverse the collateral sensitivity of resistant cells to tamoxifen. By contrast, the presence of rotenone (specific inhibitor of mitochondria complex I) synergizes with tamoxifen.ConclusionThis study demonstrates the use of tamoxifen as collateral sensitivity drug that can preferentially target multidrug resistant cells expressing P-gp at clinically achievable concentrations. Given the widespread use of tamoxifen in the treatment of estrogen receptor-positive breast cancers, this property of tamoxifen may have clinical applications in treatment of P-gp-positive drug resistant breast tumors.Graphical

Identification of bicyclic compounds that act as dual inhibitors of Bcl-2 and Mcl-1.

Elevated expression of anti-apoptotic proteins, such as Bcl-2 and Mcl-1 contributes to poor prognosis and resistance to current treatment modalities in multiple cancers. Here, we report the design, synthesis and characterization of benzimidazole chalcone and flavonoid scaffold-derived bicyclic compounds targeting both Bcl-2 and Mcl-1 by optimizing the structural differences in the binding sites of both these proteins. Initial docking screen of Bcl-2 and Mcl-1 with pro-apoptotic protein Bim revealed possible hits with optimal binding energies. All the optimized bicyclic compounds were screened for their in vitro cytotoxic activity against two oral cancer cell lines (AW8507 and AW13516) which express high levels of Bcl-2 and Mcl-1. Compound 4d from the benzimidazole chalcone series and compound 6d from the flavonoid series exhibited significant cytotoxic activity (IC50 7.12μM and 17.18μM, respectively) against AW13516 cell line. Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) analysis further demonstrated that compound 4d and compound 6d could effectively inhibit the Bcl-2 and Mcl-1 proteins by displacing their BH3 binding partners. Both compounds exhibited potent activation of canonical pathway of apoptosis evident from appearance of cleaved Caspase-3 and PARP. Further, treatment of oral cancer cells with the inhibitors induced dissociation of the BH3 only protein Bim from Mcl-1 and Bak from Bcl-2 but failed to release Bax from Bcl-xL thereby confirming the nature of compounds as BH3-mimetics selectively targeting Bcl-2 and Mcl-1. Our study thus identifies bicyclic compounds as promising candidates for anti-apoptotic Bcl-2/Mcl-1 dual inhibitors with a potential for further development.

Can the Gene Expression Profile of Patients with Acute Myeloid Leukemia Predict Complete Remission Following Induction Therapy?

Recent advances in sequencing technologies and genomics have led to the development of several targeted therapies such as BCL2 and Bromodomain and extra-terminal (BET) protein inhibitors for a more personalized treatment of patients with acute myeloid leukemia (AML), yet the majority of patients still receive standard induction chemotherapy. The molecular profiles of patients who are likely to respond to induction therapy and novel directed therapies remain to be determined. The expression of AML-related genes that are targeted by novel therapies such as BCL2 and BRD4, as well as functionally related genes and associated epigenetic modulators (TET2, EZH2, ASXL1, MYC) were analyzed in a series of 176 consecutive AML patients at multiple points during the disease course - diagnosis (Dx), post-induction (PI), complete remission (CR) and relapse (RL) - and their relationship with clinical variables and outcome investigated. Higher TET2 expression was observed PI and at CR compared to Dx, with significantly superior TET2 expression after induction therapy in the group of patients who reached CR compared to those who did not. Thus, the upregulation of TET2 at PI may be a marker of CR in AML patients. On the other hand, cells with high levels of MYC and BCL2 may be vulnerable to BRD4 inhibition.

Homocysteine promotes cardiac fibrosis by regulating the Akt/FoxO3 pathway

BackgroundEvaluated plasma homocysteine (Hcy) is an independent risk factor for cardiac fibrosis which is a common feature of cardiovascular disease, although the mechanisms are still unclear. This study aims to explore the mechanism of Hcy-induced cardiac fibrosis.MethodsThe mRNA and protein levels of Forkhead box O3 (FoxO3) and differentiation markers were detected in primary cardiac fibroblasts (CFs) after 300 µM Hcy treatment. Scratch and transwell migration assay were used to determine the effect of Hcy on proliferation and migration in CFs. The protein levels involved in the fibrotic processes in mice fed with high methionine diet (HMD) for 4 or 8 weeks were investigated by western blot. CFs were infected with FoxO3 recombinant adenovirus to explore the potential role of FoxO3 in Hcy-induced cardiac dysfunction.ResultsHcy treatment significantly promoted the differentiation, proliferation and migration of CFs, while FoxO3 activity were decreased in CFs. In HMD hearts, the protein levels of TIMP1, Fibronectin and α-SMA were increased after 4 or 8 weeks, but the FoxO3 activity was decreased. Moreover, the HMD hearts had a higher level of Bcl2 but lower of Bax and LC3II protein. In addition, FoxO3 overexpression attenuates Hcy-induced dysfunction in CFs.ConclusionsHcy promotes myofibroblast activation and resistance to autophagy and apoptosis in CFs, and eventually results in cardiac fibrosis by regulating the Akt/FoxO3 pathway. Thus, FoxO3 is a promising therapeutic target to prevent cardiac remodeling.

Deep Profiling of Chronic Lymphocytic Leukaemia (CLL) and Healthy Immune Cells By Mass Cytometry Resolves Impacts of Venetoclax Pressure

Deep Profiling of Chronic Lymphocytic Leukaemia (CLL) and Healthy Immune Cells By Mass Cytometry Resolves Impacts of Venetoclax Pressure

PRMT5 Inhibition Promotes FOXO1 Tumor Suppressor Activity to Drive a Pro-Apoptotic Program That Creates Vulnerability to Combination Treatment with Venetoclax in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an incurable B cell malignancy, comprising 5% of non-Hodgkin lymphomas diagnosed annually. MCL is associated with a poor prognosis due to emergence of resistance to immuno-chemotherapy and targeted agents. The average overall survival of patients with MCL is 4-6 years and for the majority of patients who progress on targeted agents, survival remains at a dismal 3-8 months. There is a major unmet need to identify new therapeutic approaches that are well tolerated to improve treatment outcomes and quality of life.The type II protein arginine methyltransferase enzyme, PRMT5 is overexpressed and promotes growth and survival of MCL. Inhibition of PRMT5 with a novel, SAM-competitive class of inhibitors drives anti-tumor activity in MCL cell lines and patient derived xenograft (PDX) models derived from patients with relapse or refractory disease. Selective inhibition of PRMT5 with PRT-382 (Prelude Therapeutics) in these models and MCL cell lines leads to disruption of constitutive PI3K/AKT signaling, dephosphorylation and nuclear translocation of FOXO1, and enhanced recruitment of this tumor suppressor protein to target genes. By performing chromatin immunoprecipitation-sequencing (ChIP seq) analysis, we identified over 800 newly emerged FOXO1-bound genomic loci, including multiple pro-apoptotic BCL2 family proteins (BAX, BAK1, BIK, BBC3, BMF and NOXA1). FOXO1 localization and transcriptional differences were confirmed by ChIP PCR and RT-PCR respectively. Protein levels were measured with Western blotting. BAX was identified as the most common direct target of FOXO1-transriptional activity that was upregulated on both a transcript and protein level. This led us to hypothesize that PRMT5 inhibition could potentially drive a therapeutic vulnerability to the BCL-2 inhibitor venetoclax.Single agent and combination treatment with venetoclax and PRT382 was performed in nine MCL lines. Of the nine lines, four were considered relatively resistant to PRT-382 and five resistant to venetoclax. Synergy scores, determined from MTS assays, showed significant levels of synergy in the majority of MCL lines tested. CCMCL1 and UPN1, BCL-2 negative MCL lines, and Maver1, which is highly resistant to PRMT5i, were the only cell lines to not show synergy. The cell line with the highest levels of synergy, Z-138, expressed high levels of BCL-2 and is ibrutinib resistant. Overall, there was a strong positive correlation between BCL-2 expression and synergy score (r= -0.8956, p=0.0064).The synergy seen was confirmed to be through the intrinsic apoptotic pathway based on caspase activity. To determine a mechanism of action, BAX and BAK1 were knocked down in four cell lines, three that displayed synergy and one that was resistant. This suggests that BAX expression is essential for synergy between PRMT5 and BCL2 inhibition to occur. Knock down of BAK1, the other effector of the BCL2 family of proteins, did not show protection suggesting that BAX is necessary and sufficient for this therapeutic synergy to occur. We also determined that p53 status did not correlate to the response seen (p=0.477), supporting that this mechanism is occurring through FOXO1 transcriptional regulation.In vivo evaluation in two preclinical MCL models showed therapeutic synergy with combination venetoclax/PRT382 treatment. Mice were treated with sub-therapeutic doses of venetoclax and/or PRT382 and disease burden was assessed weekly via flow cytometry. Combination treatment with well-tolerated doses of venetoclax and PRMT5 inhibitors in the MCL in vivo models showed synergistic anti-tumor activity. Both PDX models showed an extension of life with combination treatment (P<0.001) and delayed disease progression (P<0.05). This data provides mechanistic rationale while demonstrating therapeutic synergy in this preclinical study and justifies further consideration of this combination strategy targeting PRMT5 and BCL2 in MCL in the clinical setting. DisclosuresZhang: Prelude Therapeutics: Current Employment. Vaddi: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Elemento: AstraZeneca: Research Funding; Janssen: Research Funding; Johnson and Johnson: Research Funding; One Three Biotech: Consultancy, Other: Current equity holder; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Eli Lilly: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Owkin: Consultancy, Other: Current equity holder; Champions Oncology: Consultancy. Scherle: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Paik: Forkhead BioTherapeutics: Research Funding. Baiocchi: Prelude Therapeutics: Consultancy; viracta: Consultancy, Current holder of stock options in a privately-held company; Codiak Biosciences: Research Funding; Atara Biotherapeutics: Consultancy.

BTG1 Mutation Promotes Aggressive Lymphoma Development By Lowering the Threshold to MYC Activation and Generating “Super-Competitor” B Cells

BTG1 Mutation Promotes Aggressive Lymphoma Development By Lowering the Threshold to MYC Activation and Generating “Super-Competitor” B Cells

Venetoclax imparts distinct cell death sensitivity and adaptivity patterns in T cells

BH3 mimetics are increasingly used as anti-cancer therapeutics either alone or in conjunction with other chemotherapies. However, mounting evidence has also demonstrated that BH3 mimetics modulate varied amounts of apoptotic signaling in healthy immune populations. In order to maximize their clinical potential, it will be essential to understand how BH3 mimetics affect discrete immune populations and to determine how BH3 mimetic pressure causes immune system adaptation. Here we focus on the BCL-2 specific inhibitor venetoclax (ABT-199) and its effects following short-term and long-term BCL-2 blockade on T cell subsets. Seven day “short-term” ex vivo and in vivo BCL-2 inhibition led to divergent cell death sensitivity patterns in CD8+ T cells, CD4+ T cells, and Tregs resulting in shifting of global T cell populations towards a more memory T cell state with increased expression of BCL-2, BCL-XL, and MCL-1. However, twenty-eight day “long-term” BCL-2 blockade following T cell-depleted bone marrow transplantation did not lead to changes in the global T cell landscape. Despite the lack of changes in T cell proportions, animals treated with venetoclax developed CD8+ and CD4+ T cells with high levels of BCL-2 and were more resistant to apoptotic stimuli following expansion post-transplant. Further, we demonstrate through RNA profiling that T cells adapt while under BCL-2 blockade post-transplant and develop a more activated genotype. Taken together, these data emphasize the importance of evaluating how BH3 mimetics affect the immune system in different treatment modalities and disease contexts and suggest that venetoclax should be further explored as an immunomodulatory compound.

SYST-02. DUAL INHIBITION OF BCL-2 AND EGFR IN A PRECLINICAL MODEL OF LUNG CANCER BRAIN METASTASIS

Abstract Lung cancer is the most prevalent malignancy to affect both men and women. Around 80% of all lung cancers are classified as non-small cell lung cancer (NSCLC). This subtype of lung cancer is also the most likely to metastasize to the brain. Clinically, the common treatment for NSCLC is epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), due to the high occurrence of EGFR mutations. However, the cancer cells quickly develop resistance to the EGFR TKIs. This resistance and the added difficulty of delivering drugs across the blood-tumor barrier in efficacious concentrations to treat brain lesions are important to consider when developing treatment strategies for lung cancer brain metastases. Our study utilizes a NSCLC cell line, PC-9-Br6, which was developed in our laboratory to preferentially metastasize to the brain. This cell line was demonstrated by our collaborator to express higher levels of Bcl-2 in comparison to the parental PC-9-P cell line. We hypothesized combining novel Bcl-2 inhibitors (ABT-199/ABT-263) with an EGFR inhibitor (gefitinib) would increase survival and decrease tumor burden in our clinically relevant mouse model of lung cancer brain metastases.