Inhibition Of Bcl-2 Family Proteins Research Articles

Abstract 4715: Azathioprine and Z36 induced cell death in human colorectal cancer HCT-116 and HCT-15 cells

Abstract Rationale and Objectives: Cancer accounts for a huge global health burden and is the third highest cause of death across the globe. Cancer cells are known to have higher basal reactive oxygen species (ROS) levels, owing to their higher metabolism and proliferation. Higher basal ROS in cancer cells made them susceptible to exogenous ROS stimuli that lead to cell death. Therefore, in the present study, we have analyzed the effects of Azathioprine, a glutathione (GSH) inhibitor and an intracellular ROS producer. The effect of Azathioprine was analyzed alone or in combination with a BCL-2 family protein inhibitor Z36, on human colorectal cancer cell lines HCT-116 and HCT-15. Methods: The effects of the drugs have been investigated using various methods including cell viability assay, DNA fragmentation assay, and Annexin V-FITC/PI apoptosis assay. In addition, morphological changes evident in cell death were detected by Giemsa staining. Combination dosage for drug synergistic effects was computed through the use of CompuSyn software. Western blot was performed to detect changes in the expression of cell death-related proteins following drug treatments. Results and conclusion: Azathioprine at a dose of 5 µM has been demonstrated to induce cell death in both HCT116 and HCT15 cells in a concentration and time-dependent manner. On the contrary, Z36, a novel BCLXL inhibitor, has demonstrated similar cell viability results at an even lower dose of 2.5 µM. When both drugs were combined at lower doses, no significant effects were observed on the cell viability of either cell line, demonstrating that the combined treatment has no synergistic or additive effects. This suggests that both drugs do not augment each other and might induce cell death via upregulation of ROS using a similar mechanism. Citation Format: Yousuf Tahir Ali, Mahwish Fatima, Muhammad Areeb Ahmed, Hasan Salman Siddiqi, Azhar Hussain, Mati Ur Rehman. Azathioprine and Z36 induced cell death in human colorectal cancer HCT-116 and HCT-15 cells [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 4715.

Clinical trials of R-(-)-gossypol (AT-101) in newly diagnosed and recurrent glioblastoma: NABTT 0602 and NABTT 0702.

AT-101 is an oral bcl-2 family protein inhibitor (Bcl-2, Bcl-XL, Mcl-1, Bcl-W) and potent inducer of proapoptotic proteins. A prior study of the parent compound, racemic gossypol, demonstrated objective and durable responses in patients with malignant glioma. AT-101 has demonstrated synergy with radiation in animal models. The objectives of trial NABTT 0602 were to determine the MTD of AT-101 concurrent with temozolomide (TMZ) and radiation therapy (RT) (Arm I) and to determine the MTD of AT-101 when given with adjuvant TMZ after completion of standard chemoradiation (Arm 2). Separately in trial NABTT 0702, the survival and response rates of single agent AT-101 were evaluated in patients with recurrent glioblastoma. In NABTT 0602 Phase I, a 3+3 design was used to define MTDs after maximal safe resection, patients with newly diagnosed glioblastoma received standard concurrent RT (60 Gy) and TMZ 75 mg/m2/day followed by adjuvant TMZ 150-200 mg/m2 days 1-5 in 28-day cycles (Stupp regimen). In Arm I, AT-101 was administered M-F during the six weeks of RT beginning 20 mg qd. In Arm 2, concurrent with each adjuvant cycle of TMZ, AT-101 was administered at a starting dose of 20 mg, days 1-21 followed by 7-day break for a maximum of 6 cycles. The PK blood samples were collected in the first three patients in each cohort of arm 1. In NABTT 0702 patients with recurrent glioblastoma received 20 mg p.o. per day for 21 of 28 days in repeated cycles to assess overall survival (OS). A total of sixteen patients were enrolled on the two study arms of NABTT 0602. In Arm 1 AT-101 was escalated from 20 to 30 mg where one of six patients experienced DLT (grade 3 GI ulcer). On Arm 2 one patient treated at 20 mg experienced DLT (grade 3 ileus, nausea and diarrhea). The cohort was expanded to include seven patients without observation of DLT. PK results were consistent with drug levels from non-CNS studies. At study closure six patients are still alive. The median survival times for Arm I and Arm II are 15.2 months and 18.2 months, respectively. In NABTT 0702 fifty-six patients were enrolled and forty-three were eligible for imaging response. Sixteen patients (29%) had stable disease as best response and one partial response was observed. The median OS with single agent AT-101 was 5.7 months (95%CI: 3.8-7.6 months) for patients with rGBM. AT-101 can be safely administered with radiation therapy and TMZ in patients with newly diagnosed glioblastoma without toxicity unique to patients with CNS tumors. Because of toxicity observed in non-CNS AT-101 clinical trials, further dose-escalation was not attempted. The recommended dose for future studies that utilize continual AT-101 exposure is 20 mg days M-F concurrent with RT/TMZ and 20 mg days 1-21 for each 28-day cycle of TMZ. AT-101 has limited activity as a single agent in unselected patients with recurrent glioblastoma. Future trials should attempt to better understand resistance mechanisms and consider combination therapy.

Mechanisms of γδ T cell accumulation in visceral adipose tissue with aging.

γδ T cells are resident in visceral adipose tissue (VAT) where they show an age-associated increase in numbers and contribute to local and systemic chronic inflammation. However, regulation of this population and mechanisms for the age-dependent accumulation are not known. In this study, we identified a progressive trend of γδ T cell accumulation in VAT over the lifespan in mice and explored physiological mechanisms contributing to accumulation. Using isochronic parabiotic pairs of wild-type (WT) and T cell receptor delta knockout (TCRδ KO) mice at young and old age, we confirmed that VAT γδ T cells are predominately a tissue-resident population which is sustained in aging. Migration of peripheral γδ T cells into VAT was observed at less than 10%, with a decreasing trend by aging, suggesting a minor contribution of recruitment to γδ T cell accumulation with aging. Since tissue-resident T cell numbers are tightly regulated by a balance between proliferation and programmed cell death, we further explored these processes. Using in vivo EdU incorporation and the proliferation marker Ki67, we found that the absolute number of proliferating γδ T cells in VAT is significantly higher in the aged compared to young and middle-aged mice, despite a decline in the proportion of proliferating to non-proliferating cells by age. Analysis of apoptosis via caspase 3/7 activation revealed that VAT γδ T cells show reduced apoptosis starting at middle age and continuing into old age. Further, induction of apoptosis using pharmacological inhibitors of Bcl2 family proteins revealed that VAT γδ T cells at middle age are uniquely protected from apoptosis via a mechanism independent of traditional anti-apoptotic Bcl2-family proteins. Collectively, these data indicate that protection from apoptosis at middle age increases survival of tissue-resident γδ T cells resulting in an increased number of proliferative cells from middle age onward, and leading to the age-associated accumulation of γδ T cells in VAT. These findings are important to better understand how adipose tissue dysfunction and related changes in the immune profile contribute to inflammaging among the elderly.

Identification of vulnerabilities for targeting BCL-2 family members in T-Cell Acute Lymphoblastic Leukemia

T-Cell acute lymphoblastic leukemia (T-ALL) is a disease caused by the malignant transformation of T-cell lineage progenitors. With the use of intensive chemotherapies survival rates have improved, but outcomes particularly of relapsed patients remain poor. In addition, currently used intensive chemotherapies are associated with high rates of treatment-related morbidity and mortality, emphasizing the need to develop new and improved therapies. The intrinsic apoptosis pathway, one of the key pathways controlling cell death, is dysregulated in many cancers and contributes to leukemogenesis and treatment failure. The main steps of this pathway are controlled by proteins of the B-cell lymphoma (BCL-2) family at the outer mitochondrial membrane. Hence, targeting this pathway by BH3-mimetics has emerged as an effective new treatment strategy in different cancers. Venetoclax is a selective BCL-2 inhibitor and is successfully used in CLL and AML, but heterogeneous sensitivity to venetoclax has been described in ALL and inhibitors of other BCL-2 family members including BCL-XL-selective A-1331852 and MCL-1 selective AZD5991 have been developed. Moreover, a dual inhibitor of the anti-apoptotic BCL-2 family members BCL-2 and BCL-XL (AZD4320) with its dendrimer conjugate (AZD0466) has recently shown anti-tumor activity in hematologic cancer models with manageable toxicity. The aims of this study are to evaluate susceptibilities of T-ALL to inhibitors of BCL-2 family proteins, to identify markers of response, to elucidate mechanisms of action and to assess combination effects. First, we analyzed the anti-leukemia activities of inhibitors targeting BCL-2 (venetoclax), BCL-XL (A-1331852) and MCL-1 (AZD5991) and of the dual BCL-2/BCL-XL inhibitor AZD4320 in T-ALL cell lines (N=6) and a series of patient-derived xenograft (PDX) samples (N=8). Inhibition of MCL-1 alone was not effective in the cell lines tested (EC50 >1 µM), but heterogeneous sensitivity was found in PDX samples (EC50 <1µM in 3/8 samples). In cell lines, sensitivity for BCL-2 inhibition was exclusively found in a cell line with an early T-cell precursor ALL phenotype (Loucy). Similar to cell lines, we found insensitivity for BCL-2 inhibition in all PDX samples tested (N=8). For inhibition of BCL-XL, we found heterogeneous sensitivities with 4/6 cell lines and 5/8 PDX samples showing an EC 50 <1 µM. Interestingly, BH3-profiling confirmed BCL-2 dependence in Loucy and BCL-XL dependence in the other cell lines. Importantly, dual inhibition of BCL-2 and BCL-XL showed clear responses in cell lines and PDX samples (median EC 50s 796.1 nM and 721.6 nM). Notably, sensitivity was associated with priming of the BH3-peptide BAD (r s=0.89, p=0.03), providing a marker of response. We next sought to elucidate the molecular mechanisms by which different BH3-mimetics induce cell death in T-ALL. Analyzing protein interactions of BCL-2 family members by co-immunoprecipitation studies, we found that AZD4230 acts on-target by releasing BIM from BCL-2 and BCL-XL, but with compensatory increased protein complexes of BIM and MCL-1. Accordingly, combined inhibition of BCL-2 and BCL-XL with inhibition of MCL-1 leads to release of BIM from all three anti-apoptotic proteins enabling downstream apoptosis signaling. These findings suggest that combined inhibition of BCL-2 and BCL-XL together with MCL-1 may provide synergistic benefits. To further validate these data, we applied dynamic BH3-profiling and determined dependencies of T-ALL samples on BCL-2 family members in response to BCL-2/BCL-XL inhibition. Here, we found that the MCL-1 dependence strongly increases upon BCL-2/BCL-XL inhibition, suggesting MCL-1 as a target for combined inhibition. Based on these results, we performed dose-response matrix analyses to test the effects of combined BCL-2/BCL-XL and MCL-1 inhibition. Most importantly, combined inhibition showed synergism in all cell lines and PDX samples tested (Bliss scores >10), including leukemias being insensitive to single compounds. Taken together, we found vulnerabilities of T-ALL to BCL-2 family inhibition, particularly to dual BCL-2/BCL-XL inhibition by AZD4320, and we demonstrated on-target activities. Using BH3-profiling, we identified BAD-priming as a marker of response for AZD4320 and MCL-1 dependence as a resistance mechanism that can be targeted by combination treatment, suggesting further clinical evaluation.

A Phase Ib/II Study Evaluating Navitoclax after Failure of Hypomethylating Agent and Venetoclax for Treatment of Relapsed or Refractory High-Risk Myelodysplastic Syndrome

BACKGROUND AND SIGNIFICANCE Myelodysplastic syndrome (MDS) is a heterogeneous group of blood cancers of myeloid progenitor cells. They vary widely in genetic landscape, risk of progression to acute myeloid leukemia, and risk of death, as predicted by the revised international prognostic score (IPSS-R). The hypomethylating agents (HMAs) azacitidine (AZA) and decitabine (DEC) are the standard of care for treatment of high risk (HR)-MDS, however complete response rates are <15% and most patients (pts) eventually progress. Outcomes after HMA failure are dismal. Defective apoptosis is a major feature of MDS. Increased expression of anti-apoptotic members of the BCL-2 family (BCL-2, BCL-X L and BCL-W) correlates with disease progression and poor outcomes. The addition of venetoclax (VEN), a selective BCL-2 inhibitor, has shown benefit when added to AZA in pts with relapsed or refractory (R/R) MDS (Zeidan, et al., AJH, 2023). However, patterns of BCL-2 family proteins are heterogeneous and response to inhibition of BCL-2 family proteins depends on the balance of pro- and anti-apoptotic proteins. Navitoclax (NAV) is a broad oral inhibitor of BCL-2, BCL-X L, and BCL-W. Increased expression of BCL-X L has been shown to make MDS cells insensitive to VEN. Further, VEN-resistant myeloid cell lines derived by prolonged VEN exposure showed up-regulation of BCL-X L, driving acquired drug resistance in these cell lines. Thus, the addition of NAV to the combination of VEN and HMA is a logical strategy to induce response in pts with R/R after HMA with or without prior VEN exposure. STUDY DESIGN AND METHODS We will perform a phase Ib/II, dose escalation and expansion study to evaluate escalating doses of NAV in combination with standard dose VEN and DEC in pts with R/R HR-MDS after failure of HMA (NCT05564650). The primary objective of the phase Ib portion of the study will be to evaluate the safety profile and determine the recommended phase II dose (RP2D) of NAV in the combination. The primary objective of the phase II portion of the study will be to evaluate the efficacy of combination therapy NAV/VEN/DEC in pts with R/R HR-MDS after failure of HMA/VEN. During cycle 1, to limit the risk of tumor lysis, pts will be treated with a short dose ramp-up with VEN 200 mg daily on day 1 and 400 mg daily on days 2-16, DEC 20 mg/m2 on days 3-7 and NAV 25 mg, 50 mg or 100 mg daily on days 3-16 during cycle 1. During cycles 2 and beyond, dosing will be similar to cycle 1 without dose ramp-up ( Figure 1). We will employ the Bayesian optimal interval (BOIN) design to identify the maximum tolerated dose and RP2D of NAV. Subsequently, the study will proceed to phase II to further evaluate the safety and efficacy of the combination. The study will enroll pts with IPSS-R ≥3 who have been previously treated with HMA/VEN. Pts in the phase I portion of the trial may be enrolled if they have not received VEN. Pts must have adequate performance status and organ function. Pts with a WBC >25,000 cells/µL and those taking prohibited medications will be excluded. Correlative studies will be performed to determine the impact of treatment on molecular targets and will include profiling of BCL-2 family members and single cell gene expression patterns pre- and post-treatment and at relapse. Pts who achieve a complete response (CR) or modified CR (mCR) will continue on the study until disease progression, dose limiting toxicity, availability of an alternative therapy or withdrawal of consent. Pts who have hematologic improvement but who do not attain CR or mCR can continue the study at the discretion of their treating physician in conjunction with the primary investigator.

ASXL1 mutations are associated with a response to alvocidib and 5-azacytidine combination in myelodysplastic neoplasms.

Inhibitors of anti-apoptotic BCL-2 family proteins in combination with chemotherapy and hypomethylating agents (HMA) are promising therapeutic approaches in acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS). Alvocidib, a cyclin-dependent kinase 9 (CDK9) inhibitor and indirect transcriptional repressor of the anti-apoptotic factor MCL-1, has previously shown clinical activity in AML. Availability of biomarkers for response to the alvocidib + 5-azacytidine (5-AZA) could also extend the rationale of this treatment concept to high-risk MDS. In this study, we performed a comprehensive in vitro assessment of alvocidib and 5-AZA effects in N=45 high-risk MDS patients. Our data revealed additive cytotoxic effects of the combination treatment. Mutational profiling of MDS samples identified ASXL1 mutations as predictors of response. Further, increased response rates were associated with higher gene expression of the pro-apoptotic factor NOXA in ASXL1-mutated samples. The higher sensitivity of ASXL1 mutant cells to the combination treatment was confirmed in vivo in ASXL1Y588X transgenic mice. Overall, our study demonstrated augmented activity for the alvocidib + 5-AZA combination in higher-risk MDS and identified ASXL1 mutations as a biomarker of response for potential stratification studies.

Developing 3D Organoid Raft Cultures from Patient-Derived Xenografts as Rapid Models to Screen Efficacy of Experimental Therapeutics.

Reliable preclinical models are needed for screening new cancer drugs. Thus, we developed an improved 3D tumor organoid model termed "organoid raft cultures" (ORCs). Development of ORCs involved culturing tumors ex vivo on collagen beds (boats) with grid supports to maintain their morphological structure. The ORCs were developed from patient-derived xenografts (PDXs) of colon cancers excised from immune-deficient mice (NOD/SCID/IL2Rgammanull). We utilized these new models to evaluate the efficacy of an investigational drug, Navitoclax (ABT-263). We tested the efficacy of ABT-263, an inhibitor of BCL-2 family proteins, in these ORCs derived from a PDX that showed high expression of antiapoptotic BCL2 family proteins (BCL-2, BCL-XL, and BCL-W). Hematoxylin and eosin staining evaluation of PDXs and corresponding ORCs indicated the retention of morphological and other histological integrity of ORCs. ORCs treated with ABT-263 showed decreased expression of antiapoptotic proteins (BCL2, BCL-XL and BCL-W) and increased proapoptotic proteins (BAX and PUMA), with concomitant activation of caspase 3. These studies support the usefulness of the ORCs, developed from PDXs, as an alternative to PDXs and as faster screening models.

ASXL1 Mutations Are Associated with a Response to the Combination of Alvocidib and 5-Azacytidine in Higher-Risk Myelodysplastic Syndromes

Introduction: Inhibitors of anti-apoptotic BCL-2 family proteins in combination with chemotherapy and hypomethylating agents (HMAs) are perspective therapeutic approaches in acute myeloid leukemia (AML) and higher-risk myelodysplastic syndromes (MDS). Inhibitors of BCL-2 family proteins previously showed potent activity against AML and higher-risk MDS. Alvocidib (Alv), a cyclin-dependent kinase 9 inhibitor and indirect transcriptional repressor of the anti-apoptotic BCL-2 family member MCL-1, has previously shown clinical activity in AML (Lee DJ et al, Expert Opin Investig Drugs 2019; Zeidner JF et al, Leuk Res 2015). A phase 1b/2 study with Alv and HMAs 5-Azacytidine (5-AZA) or decitabine in higher-risk MDS patients was recently completed (NCT03593915). However, biomarkers of response to 5-AZA + Alv combination are insufficiently characterized. In order to possibly identify biomarkers of response, we performed a comprehensive in vitro assessment of Alv and 5-AZA effects in a cohort of n=45 higher-risk MDS patients. Methods: CD34+ hematopoietic stem and progenitor cells (HSPCs) were isolated from bone marrow (BM) aspirates using positive selection with MACS microbeads. CD34+ cells of aged-matched healthy controls (HC, n=11) were obtained from bone specimen after femur endoprosthesis surgery. HSPCs were expanded for four days in StemSpan SFEM II medium containing StemSpan Myeloid Expansion Supplement (Stem Cell Technologies) and treated with 5-AZA for 48h, Alv for 24h or their sequential combination (5-AZA for 48h followed by Alv for 24h). Cell viability was determined using CellTiter-Glo (CTG) and Annexin-V apoptosis assays. MCL-1 dependency of primary MDS HSPCs was assessed using NOXA-derived T-MS1 peptide. MDS recurrent mutations in BM mononuclear cells were assessed using myeloid NGS panel deep sequencing containing 67 genes. For in vivo tumor transfer experiments, splenic cells from CD45.2+Asxl1Y588X transgenic (Tg) mice were injected intravenously into sublethally irradiated CD45.1+ recipients through tail veins. Engrafted mice were treated with 1 mg/kg 5-AZA intraperitoneally (i.p.) on days 1-3 (qdx) every other week. Alv was administered i.p. at a dose of 2.5 mg/kg on day 5 (qw) in the weeks when 5-AZA was applied. Mice were sacrificed 11 weeks after the transplantation and the percentage of CD45.2+ cells in the BM was assessed using flow cytometric analysis. Results: The combination of 5-AZA+Alv showed an additive cytotoxic effect on MDS cells in CTG cell viability assays (median cell viability was 74%, 74% and 55% for 5-AZA, Alv and combination, respectively, p<0.0001). In annexin-V apoptotic assays, MDS cells were more sensitive to the cytotoxic effect of the combination treatment compared to HC (median percent of apoptotic and dead cells was 37% for MDS vs 26% for HC, p=0.0256). In analogy to in vitro studies, accelerated clearance of xenografted MDS-L cells from the peripheral blood of NSGS mice was observed after 5-AZA+Alv treatment compared to single agents. MCL-1 dependency of primary MDS samples did not significantly correlate with cytotoxic effects of Alv and combination treatment. However, the presence of ASXL1 mutations was significantly associated with higher cytotoxic activity of 5-AZA+Alv combination in univariable (p=0.0044) and multivariable (p=0.042) analyses. Increased sensitivity of BM cells to the cytotoxic effects of 5-AZA+Alv was also observed in Asxl1Y588XTg mice. The combination treatment facilitated clearance of CD45.2+Asxl1Y588X mutant leukemic cells from CD45.1+ recipients compared to 5-AZA monotherapy (p=0.0156). Histological analysis of BM cells after 5-AZA+Alv treatment in CD45.1+ recipients showed decrease in the percentage of immature blasts and improved development of mature BM cells when compared to 5-AZA monotherapy (p=0.0391). Improved cytotoxic effects of Alv and 5-AZA+Alv in ASXL1 mutant compared to ASXL1 wild type MDS samples were associated with higher expression of pro-apoptotic MCL-1 antagonist NOXA (p=0.0017). Conclusions: We provided a pre-clinical rationale for the use of the 5-AZA+Alv combination for higher risks MDS and proposed ASXL1 mutations as potential genetic biomarkers of response in prospective clinical trials.

EXTH-03. HSP90 INHIBITION OVERCOMES RESISTANCE TO MOLECULAR TARGETED THERAPY IN BRAFV600E MUTANT HIGH-GRADE GLIOMA

Abstract INTRODUCTION Molecular targeted therapy using BRAF and/or MEK inhibitors has been applied to BRAFV600E mutant high-grade gliomas (HGGs); however, the therapeutic effect is limited by the emergence of drug resistance. METHODS We established multiple paired BRAFV600E mutant HGG patient-derived xenograft (PDX) models based on tissues collected prior to and at relapse after molecular targeted therapy. Using these models, we dissected treatment resistant mechanisms for molecular targeted therapy and explored therapeutic targets to overcome resistance in BRAFV600E HGG models in vitro and in vivo. RESULTS We found that, despite causing no major genetic and epigenetic changes, BRAF and/or MEK inhibitor treatment deregulated multiple negative feedback mechanisms, which led to the re-activation of the MAPK pathway through c-Raf and AKT signaling. This altered oncogenic signaling primarily mediated resistance to molecular targeted therapy in BRAFV600E mutant HGG. To overcome this resistance mechanism, we performed a high-throughput drug screening to identify therapeutic agents that potently induce additive cytotoxicity with BRAF and MEK inhibitors. We discovered that HSP90 inhibition combined with BRAF/MEK inhibition coordinately deactivated the MAPK and AKT/mTOR pathways, and subsequently induced apoptosis via dephosphorylation of GSK3β (Ser9) and inhibition of Bcl-2 family proteins. This mediated potent cytotoxicity in vitro and in vivo in refractory models with acquired resistance to molecular-targeted therapy. CONCLUSIONS The combination of an HSP90 inhibitor with BRAF or MEK inhibitors can overcome the limitations of the current therapeutic strategies for BRAFV600E mutant HGG.

The Increase in the Drug Resistance of Acute Myeloid Leukemia THP-1 Cells in High-Density Cell Culture Is Associated with Inflammatory-like Activation and Anti-Apoptotic Bcl-2 Proteins.

It is known that cell culture density can modulate the drug resistance of acute myeloid leukemia (AML) cells. In this work, we studied the drug sensitivity of AML cells in high-density cell cultures (cell lines THP-1, HL-60, MV4-11, and U937). It was shown that the AML cells in high-density cell cultures in vitro were significantly more resistant to DNA-damaging drugs and recombinant ligand izTRAIL than those in low-density cell cultures. To elucidate the mechanism of the increased drug resistance of AML cells in high-density cell cultures, we studied the activation of Bcl-2, Hif-1alpha, and NF-kB proteins, as well as cytokine secretion, the inflammatory immunophenotype, and the transcriptome for THP-1 cells in the low-density and high-density cultures. The results indicated that the increase in the drug resistance of proliferating THP-1 cells in high-density cell cultures was associated with the accumulation of inflammatory cytokines in extracellular medium, and the formation of NF-kB-dependent inflammatory-like cell activation with the anti-apoptotic proteins Bcl-2 and Bcl-xl. The increased drug resistance of THP-1 cells in high-density cultures can be reduced by ABT-737, an inhibitor of Bcl-2 family proteins, and by inhibitors of NF-kB. The results suggest a mechanism for increasing the drug resistance of AML cells in the bone marrow and are of interest for developing a strategy to suppress this resistance.

Gemcitabine Cooperates with Everolimus to Inhibit the Growth of and Sensitize Malignant Meningioma Cells to Apoptosis Induced by Navitoclax, an Inhibitor of Anti-Apoptotic BCL-2 Family Proteins.

Simple SummaryMeningioma is the most common intracranial neoplasm derived from the arachnoid cap cells of the leptomeninges. Malignant meningioma is generally more aggressive than other meningioma and frequently recurs even after surgery and radiation therapy. Clinical trials have been performed on candidate drugs, including everolimus, an inhibitor of mammalian target of rapamycin. However, an effective standard systemic therapy has not yet been established and the prognosis of patients with malignant meningioma is still poor. We recently reported the radiosensitization effects of gemcitabine in malignant meningioma cells, which suggests its potential to enhance the efficacy of candidate drugs for meningioma. In the present study, we demonstrated that gemcitabine enhanced the therapeutic effects of everolimus in malignant meningioma cells, and these effects were further augmented by navitoclax, an inhibitor of anti-apoptotic BCL-2 family proteins, both in vitro and in vivo. The present results provide support for the clinical application of gemcitabine and navitoclax in combination with everolimus to the treatment of patients with malignant meningioma.Despite several clinical trials with encouraging findings, effective standard systemic therapies have yet to be established for malignant meningioma and the prognosis of these patients remains poor. Accumulating preclinical and clinical evidence suggests that gemcitabine is effective against malignant meningioma. To identify drugs with therapeutic effects that may be enhanced in combination with gemcitabine, we screened drugs that have been tested in preclinical and clinical trials for meningioma. In IOMM-Lee and HKBMM malignant meningioma cells, gemcitabine enhanced the growth inhibitory effects of the mTOR inhibitor everolimus, the clinical benefits of which have been demonstrated in patients with meningioma. The synergistic growth inhibitory effects of this combination were accompanied by cellular senescence characterized by an increase in senescence-associated β-galactosidase activity. To enhance the effects of this combination, we screened senolytic drugs that selectively kill senescent cells, and found that navitoclax, an inhibitor of anti-apoptotic BCL-2 family proteins, effectively reduced the number of viable malignant meningioma cells in combination with everolimus and gemcitabine by inducing apoptotic cell death. The suppression of tumor growth in vivo by the combination of everolimus with gemcitabine was significantly stronger than that by either treatment alone. Moreover, navitoclax, in combination with everolimus and gemcitabine, significantly reduced tumor sizes with an increase in the number of cleaved caspase-3-positive apoptotic cells. The present results suggest that the addition of gemcitabine with or without navitoclax to everolimus is a promising strategy that warrants further evaluation in future clinical trials for malignant meningioma.

HSP90 Inhibition Overcomes Resistance to Molecular Targeted Therapy in BRAFV600E-mutant High-grade Glioma.

Molecular targeted therapy using BRAF and/or MEK inhibitors has been applied to BRAFV600E-mutant high-grade gliomas (HGG); however, the therapeutic effect is limited by the emergence of drug resistance. We established multiple paired BRAFV600E-mutant HGG patient-derived xenograft models based on tissues collected prior to and at relapse after molecular targeted therapy. Using these models, we dissected treatment-resistant mechanisms for molecular targeted therapy and explored therapeutic targets to overcome resistance in BRAFV600E HGG models in vitro and in vivo. We found that, despite causing no major genetic and epigenetic changes, BRAF and/or MEK inhibitor treatment deregulated multiple negative feedback mechanisms, which led to the reactivation of the MAPK pathway through c-Raf and AKT signaling. This altered oncogenic signaling primarily mediated resistance to molecular targeted therapy in BRAFV600E-mutant HGG. To overcome this resistance mechanism, we performed a high-throughput drug screening to identify therapeutic agents that potently induce additive cytotoxicity with BRAF and MEK inhibitors. We discovered that HSP90 inhibition combined with BRAF/MEK inhibition coordinately deactivated the MAPK and AKT/mTOR pathways, and subsequently induced apoptosis via dephosphorylation of GSK3β (Ser9) and inhibition of Bcl-2 family proteins. This mediated potent cytotoxicity in vitro and in vivo in refractory models with acquired resistance to molecular targeted therapy. The combination of an HSP90 inhibitor with BRAF or MEK inhibitors can overcome the limitations of the current therapeutic strategies for BRAFV600E-mutant HGG.

Scaffold hopping from indoles to indazoles yields dual MCL-1/BCL-2 inhibitors from MCL-1 selective leads.

Overexpression of the anti-apoptotic BCL-2 proteins is associated with the development and progression of a range of cancers. Venetoclax, an FDA-approved BCL-2 inhibitor, is fast becoming the standard-of-care for acute myeloid leukemia and chronic lymphocytic leukemia. However, the median survival offered by venetoclax is only 18 months (as part of a combination therapy regimen), and one of the primary culprits for this is the concomitant upregulation of sister anti-apoptotic proteins, in particular MCL-1 (and BCL-xL), which provides an escape route that manifests as venetoclax resistance. Since inhibition of BCL-xL leads to thrombocytopenia, we believe that a dual MCL-1/BCL-2 inhibitor may provide an enhanced therapeutic effect relative to a selective BCL-2 inhibitor. Beginning with a carboxylic acid-containing literature compound that is a potent inhibitor of MCL-1 and a moderate inhibitor of BCL-2, we herein describe our efforts to develop dual inhibitors of MCL-1 and BCL-2 by scaffold hopping from an indole core to an indazole framework. Subsequently, further elaboration of our novel N2-substituted, indazole-3-carboxylic acid lead into a family of indazole-3-acylsulfonamides resulted in improved inhibition of both MCL-1 and BCL-2, possibly through occupation of the p4 pocket, with minimal or no inhibition of BCL-xL.

Inhibitors of Anti-apoptotic Bcl-2 Family Proteins Exhibit Potent and Broad-Spectrum Anti-mammarenavirus Activity via Cell Cycle Arrest at G0/G1 Phase.

ABSTRACTTargeting host factors is a promising strategy to develop broad-spectrum antiviral drugs. Drugs targeting anti-apoptotic Bcl-2 family proteins that were originally developed as tumor suppressors have been reported to inhibit multiplication of different types of viruses. However, the mechanisms whereby Bcl-2 inhibitors exert their antiviral activity remain poorly understood. In this study, we have investigated the mechanisms by which obatoclax (OLX) and ABT-737 Bcl-2 inhibitors exhibited a potent antiviral activity against the mammarenavirus lymphocytic choriomeningitis virus (LCMV). OLX and ABT-737 potent anti-LCMV activity was not associated with their proapoptotic properties but rather with their ability to induce cell arrest at the G0/G1 phase. OLX- and ABT-737–mediated inhibition of Bcl-2 correlated with reduced expression levels of thymidine kinase 1 (TK1), cyclin A2 (CCNA2), and cyclin B1 (CCNB1) cell cycle regulators. In addition, small interfering RNA (siRNA)–mediated knockdown of TK1, CCNA2, and CCNB1 resulted in reduced levels of LCMV multiplication. The antiviral activity exerted by Bcl-2 inhibitors correlated with reduced levels of viral RNA synthesis at early times of infection. Importantly, ABT-737 exhibited moderate efficacy in a mouse model of LCMV infection, and Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS–CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals.IMPORTANCE Antiapoptotic Bcl-2 inhibitors have been shown to exert potent antiviral activities against various types of viruses via mechanisms that are currently poorly understood. This study has revealed that Bcl-2 inhibitors’ mediation of cell cycle arrest at the G0/G1 phase, rather than their proapoptotic activity, plays a critical role in blocking mammarenavirus multiplication in cultured cells. In addition, we show that Bcl-2 inhibitor ABT-737 exhibited moderate antimammarenavirus activity in vivo and that Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals.

Abstract 1943: Radiosensitization of PIK3CA wild type triple negative breast cancers with Bcl-family inhibition

Abstract Purpose: Compared to other breast cancer subtypes, triple negative breast cancers (TNBC) derive the least benefit from adjuvant radiation (RT) which contributes to higher rates of locoregional recurrence. Thus, there is a critical need to identify clinical strategies to increase the effectiveness of RT therapy in TNBC. Methods: Alamar blue proliferation assays were used to calculate half maximal inhibitory concentration (IC50) values for each Bcl-2 family inhibitor 72 hours after drug treatment. Clonogenic survival assays were used to evaluate radiosensitivity and to calculate the radiation enhancement ratio (rER) after combination treatment. Apoptosis was assessed through formation of cleaved PARP and annexin V/PI-based flow cytometry. Xenograft models with MDA-MB-231 cells and TNBC patient-derived xenografts (PDX4664) were used to assess radiosensitization in vivo. Results: A novel radiosensitizer screen identified Bcl-2 family inhibition as a potentially effective treatment strategy in radioresistant breast cancer models. Single-agent response to pan Bcl-2 family inhibition (ABT-263) or Bcl-xL inhibition (WEHI-539, A-1331852) was more effective in PIK3CA wild type (wt) TNBC (IC50 &amp;lt; 1µM) compared to PIK3CA mutant TNBC. Inhibition of apoptosis with ABT-263 led to radiosensitization of PIK3CA/PTEN wild-type TNBC cell lines (rER: 1.09-1.74), but had no effect on PIK3CA/PTEN mutant TNBC (rER: 0.87-1.18). Radiosensitization was observed to be Bcl-xL-dependent, with Bcl-xL inhibitor-specific radiosensitization (rER: 1.12-2.38) but a lack of Bcl-2 inhibitor (ABT-199, rER: 0.94 - 1.21) or MCL-1 inhibitor-mediated radiosensitization (S63845, rER: 0.91 - 1.06). In PIK3CA wt TNBC, combination treatment of Bcl-2 family inhibition and RT significantly increased the percent of apoptotic cells (p &amp;lt; 0.001) and led to increased formation of cleaved PARP 48 hours after RT. Sensitivity to RT was dependent on expression of MCL-1, an anti-apoptotic protein that is overexpressed in PIK3CA/PTEN mutant TNBC. Overexpression of MCL-1 in PIK3CA/PTEN wild type TNBC rescued radioresistance (rER: 0.99-1.09), whereas co-inhibition of MCL-1 and Bcl-xL in PIK3CA/PTEN mutant TNBC was sufficient to overcome radioresistance (rER: 2.32 - 2.35). In vivo, nonspecific Bcl-2 family inhibition or specific Bcl-xL inhibition in combination with RT decreased tumor growth and increased time to tumor tripling (p &amp;lt; 0.0001) in PIK3CA wt models of TNBC. Conclusions: In this study, we demonstrated that inhibition of Bcl-2 family proteins in combination with RT led to increased levels of apoptosis and cell death in PIK3CA/PTEN wt - but not PIK3CA/PTEN mutant - TNBC and we identified MCL-1 as a critical mediator of this radiosensitIvity. Together, these results indicate that Bcl-xL inhibition may be a feasible clinical strategy for the radiosensitization of PIK3CA/PTEN wild-type TNBC. Citation Format: Andrea M. Pesch, Benjamin C. Chandler, Anna R. Michmerhuizen, Nicole Hirsh, Kari Wilder-Romans, Meilan Liu, Tanner Ward, Dana Messinger, Charles Nino, Cassandra Ritter, James M. Rae, Corey W. Speers. Radiosensitization of PIK3CA wild type triple negative breast cancers with Bcl-family inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1943.

Mapping mitochondrial respiratory chain deficiencies by respirometry: Beyond the Mito Stress Test

Cell-based respirometers, such as the Seahorse Extracellular Flux Analyzer, are valuable tools to assess the functionality of mitochondria within adherent neurons, as well as other cell types. The Mito Stress Test is the most frequently employed protocol of drug additions to evaluate mitochondrial bioenergetic function. Sequential exposure of cells to an ATP synthase inhibitor such as oligomycin and an uncoupler such as FCCP cause changes in oxygen consumption rate that allow estimation of the cellular efficiency and capacity for mitochondrial ATP synthesis. While a useful first step in assessing whether an experimental treatment or genetic manipulation affects mitochondrial energetics, the Mito Stress Test does not identify specific sites of altered respiratory chain function. This article discusses limitations of the Mito Stress Test, proposes a refined protocol for comparing cell populations that requires independent drug titrations at multiple cell densities, and describes a stepwise series of respirometry-based assays that “map” locations of electron transport deficiency. These include strategies to test for cytochrome c release, to probe the functionality of specific electron transport chain complexes within intact or permeabilized cells, and to measure NADH oxidation by the linked activity of Complexes I, III, and IV. To illustrate utility, we show that although UK5099 and ABT-737 each decrease the spare respiratory capacity of cortical neurons, the stepwise assays reveal different underlying mechanisms consistent with their established drug targets: deficient Complex I substrate supply induced by the mitochondrial pyruvate carrier inhibitor UK5099 and cytochrome c release induced by the anti-apoptotic BCL-2 family protein inhibitor ABT-737.

Exploring the potential of BH3 mimetic therapy in squamous cell carcinoma of the head and neck

Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common cancer worldwide, with overall survival of less than 50%. Current therapeutic strategies involving a combination of surgery, radiation, and/or chemotherapy are associated with debilitating side effects, highlighting the need for more specific and efficacious therapies. Inhibitors of BCL-2 family proteins (BH3 mimetics) are under investigation or in clinical practice for several hematological malignancies and show promise in solid tumors. In order to explore the therapeutic potential of BH3 mimetics in the treatment of SCCHN, we assessed the expression levels of BCL-2, BCL-XL, and MCL-1 via Western blots and immunohistochemistry, in cell lines, primary cells derived from SCCHN patients and in tissue microarrays containing tumor tissue from a cohort of 191 SCCHN patients. All preclinical models exhibited moderate to high levels of BCL-XL and MCL-1, with little or no BCL-2. Although expression levels of BCL-XL and MCL-1 did not correlate with patient outcome, a combination of BH3 mimetics to target these proteins resulted in decreased clonogenic potential and enhanced apoptosis in all preclinical models, including tumor tissue resected from patients, as well as a reduction of tumor volume in a zebrafish xenograft model of SCCHN. Our results show that SCCHN is dependent on both BCL-XL and MCL-1 for apoptosis evasion and combination therapy targeting both proteins may offer significant therapeutic benefits in this disease.

EBV BCL-2 homologue BHRF1 drives chemoresistance and lymphomagenesis by inhibiting multiple cellular pro-apoptotic proteins.

Epstein-Barr virus (EBV), which is ubiquitous in the adult population, is causally associated with human malignancies. Like many infectious agents, EBV has evolved strategies to block host cell death, including through expression of viral homologues of cellular BCL-2 pro-survival proteins (vBCL-2s), such as BHRF1. Small molecule inhibitors of the cellular pro-survival BCL-2 family proteins, termed 'BH3-mimetics', have entered clinical trials for blood cancers with the BCL-2 inhibitor venetoclax already approved for treatment of therapy refractory chronic lymphocytic leukaemia and acute myeloid leukaemia in the elderly. The generation of BH3-mimetics that could specifically target vBCL-2 proteins may be an attractive therapeutic option for virus-associated cancers, since these drugs would be expected to only kill virally infected cells with only minimal side effects on normal healthy tissues. To achieve this, a better understanding of the contribution of vBCL-2 proteins to tumorigenesis and insights into their biochemical functions is needed. In the context of Burkittlymphoma (BL), BHRF1 expression conferred strong resistance to diverse apoptotic stimuli. Furthermore, BHRF1 expression in mouse haematopoietic stem and progenitor cells accelerated MYC-induced lymphoma development in a model of BL. BHRF1 interacts with the cellular pro-apoptotic BCL-2 proteins, BIM, BID, PUMA and BAK, but its capability to inhibit apoptosis could not be mapped solely to one of these interactions, suggesting plasticity is a key feature of BHRF1. Site-directed mutagenesis revealed a site in BHRF1 that was critical for its interaction with PUMA and blocking DNA-damage-induced apoptosis, identifying a potentially therapeutically targetable vulnerability in BHRF1.

DRP-1 functions independently of mitochondrial structural perturbations to facilitate BH3 mimetic-mediated apoptosis

Maintenance of mitochondrial integrity is critical for normal cellular homoeostasis. Most cells respond to stress stimuli and undergo apoptosis by perturbing mitochondrial structure and function to release proteins, such as cytochrome c, which are essential for the execution of the intrinsic apoptotic cascade. Cancer cells evade these events by overexpressing the anti-apoptotic BCL-2 family of proteins on mitochondrial membranes. Inhibitors of the anti-apoptotic BCL-2 family proteins, also known as BH3 mimetics, antagonise the pro-survival functions of these proteins and result in rapid apoptosis. Although the precise mechanism by which BH3 mimetics induce apoptosis has been well characterised, not much is known in terms of the structural changes that occur in mitochondria during apoptosis. Using a panel of highly selective BH3 mimetics and a wide range of cell lines, we demonstrate that BH3 mimetics induce extensive mitochondrial fission, accompanied by swelling of the mitochondrial matrix and rupture of the outer mitochondrial membrane. These changes occur in a BAX/ BAK-dependent manner. Although a major mitochondrial fission GTPase, DRP-1, has been implicated in mitochondrial apoptosis, our data demonstrate that DRP-1 might function independently/downstream of BH3 mimetic-mediated mitochondrial fission to facilitate the release of cytochrome c and apoptosis. Moreover, downregulation of DRP-1 prevented cytochrome c release and apoptosis even when OPA1, a protein mediating mitochondrial fusion, was silenced. Although BH3 mimetic-mediated displacement of BAK and other BH3-only proteins from BCL-XL and MCL-1 was unaffected by DRP-1 downregulation, it prevented BAK activation significantly, thus placing DRP-1 as one of the most critical players, along with BAX and BAK, that governs BH3 mimetic-mediated cytochrome c release and apoptosis.

Heme Oxygenase-1 is a Key Molecule Underlying Differential Response of TW-37-Induced Apoptosis in Human Mucoepidermoid Carcinoma Cells.

TW-37 is a small-molecule inhibitor of Bcl-2 family proteins, which can induce anti-cancer activities in various types of cancer. In the current study, we investigated the potential molecular mechanism underlying the differential response to TW-37-induced apoptosis in two human mucoepidermoid carcinoma (MEC) cell lines. The differential response and underlying molecular mechanism of human MEC cells to TW-37 was evaluated by trypan blue exclusion assay, western blotting, 4’, 6-diamidino-2-phenylindole staining, annexin V/propidium iodide double staining, analysis of the sub-G1 population, human apoptosis array, and measurements of intracellular reactive oxygen species (ROS). TW-37 decreased cell viability and induced apoptosis in YD-15 cells, but not in MC3 cells. Proteome profiling using a human apoptosis array revealed four candidate proteins and of these, heme oxygenase-1 (HO-1) was mainly related to the differential response to TW-37 of YD-15 and MC3 cells. TW-37 also led to a significant increase in intracellular levels of ROS in YD-15 cells, which is associated with apoptosis induction. The ectopic expression of HO-1 recovered YD-15 cells from TW-37-induced apoptosis by reducing intracellular levels of ROS. The expression of HO-1 was reduced through both transcriptional and post-translational modification during TW-37-mediated apoptosis. We conclude that HO-1 is a potential indicator to estimate response to TW37-induced apoptosis in human MEC.