Anti-apoptotic Protein Mcl-1 Research Articles

Antioxidant and cytotoxic activities of different solvent fractions from Murraya koenigii shoots: HPLC quantification And molecular docking of identified phenolics with anti-apoptotic proteins

ABSTRACT. Murraya koenigii is known for its health benefits against constipation, diarrhea, bacterial infections, wounds and skin related diseases. Aim of this project is to determine cytotoxic aptitude of antioxidant compounds present in M. koenigii. The fractionation of M. koenigii shoots methanol extract was carried out with different solvents followed by determination of total phenolic content, radical scavenging potential along with phenolic profile. M. koenigii shoot fractions were analyzed for their cytotoxic potential by MTT assay besides evaluating molecular interactions between identified phenolics with Bcl-2, Bcl-xl and MCL-1. The results revealed that butanol fraction contains maximum amount of quercetin, 4-hydroxy-3-methoxy benzoic acid and trans-4-hydroxy-3-methoxy cinnamic acid. Ferulic acid is abundant in water fraction whereas n-hexane fractions contain sinapic and vanillic acids. The ethyl acetate fraction possess the highest level of phenolics as well as radical scavenging potential. HPLC results show that 9 organic acids are present in ethyl acetate and butanol fractions. The highest cytotoxic activity was exhibited by n-hexane and ethyl acetate fractions. Molecular docking studies supports that ethyl acetate and n-hexane fractions are the major sources of antioxidant and cytotoxic compounds. Also, molecular interactions exist between identified phenolics from plant shoots fractions with anti-apoptotic proteins Bcl-2, Bcl-xl and MCL-1.
 
 KEY WORDS: Morraya koenigii, Fractionation, Antioxidant, Cytotoxic, Molecular docking
 Bull. Chem. Soc. Ethiop. 2022, 36(3), 651-666. 
 DOI: https://dx.doi.org/10.4314/bcse.v36i3.14

Diverse actions of sirtuin-1 on ovulatory genes and cell death pathways in human granulosa cells

BackgroundHuman granulosa-lutein cells (hGLCs) amply express sirtuin-1 (SIRT1), a NAD + -dependent deacetylase that is associated with various cellular functions. SIRT1 was shown to elevate cAMP on its own and additively with human chorionic gonadotropin (hCG), it is therefore interesting to examine if SIRT1 affects other essential hGLC functions.MethodsPrimary hGLCs, obtained from the follicular aspirates of women undergoing IVF and SV40-transfected, immortalized hGLCs (SVOG cells), were used. Primary cells were treated with SIRT1 specific activator SRT2104, as well as hCG or their combination. Additionally, siRNA-targeting SIRT1 construct was used to silence endogenous SIRT1 in SVOG cells. PTGS2, EREG, VEGFA and FGF2 expression was determined using quantitative polymerase chain reaction (qPCR). Apoptotic and necroptotic proteins were determined by specific antibodies in western blotting. Cell viability/apoptosis was determined by the XTT and flow cytometry analyses. Data were analyzed using student t-test or Mann–Whitney U test or one-way ANOVA followed by Tukey HSD post hoc test.ResultsIn primary and immortalized hGLCs, SRT2104 significantly upregulated key ovulatory and angiogenic genes: PTGS2, EREG, FGF2 and VEGFA, these effects tended to be further augmented in the presence of hCG. Additionally, SRT2104 dose and time-dependently decreased viable cell numbers. Flow cytometry of Annexin V stained cells confirmed that SIRT1 reduced live cell numbers and increased late apoptotic and necrotic cells. Moreover, we found that SIRT1 markedly reduced anti-apoptotic BCL-XL and MCL1 protein levels and increased cleaved forms of pro-apoptotic proteins caspase-3 and PARP. SIRT1 also significantly induced necroptotic proteins RIPK1 and MLKL. RIPK1 inhibitor, necrostatin-1 mitigated SIRT1 actions on RIPK1 and MLKL but also on cleaved caspase-3 and PARP and in accordance on live and apoptotic cells, implying a role for RIPK1 in SIRT1-induced cell death. SIRT1 silencing produced inverse effects on sorted cell populations, anti-apoptotic, pro-apoptotic and necroptotic proteins, corroborating SIRT1 activation.ConclusionsThese findings reveal that in hGLCs, SIRT1 enhances the expression of ovulatory and angiogenic genes while eventually advancing cell death pathways. Interestingly, these seemingly contradictory events may have occurred in a cAMP-dependent manner.

Synthesis, Anticancer Evaluation and Molecular Docking of Hexahydroquinoline Derivatives as Mcl-1 Inhibitors and Apoptosis Inducers.

Hexahydroquinoline as a small molecule was reported for good cytotoxicity and affinity towards Mcl-1. Hence, new compounds were explored as Mcl-1 inhibitors to be potent anticancer agents. Compounds were synthesized and screened for cytotoxicity. The active compound was evaluated for cell cycle analysis, Mcl-1 inhibition, caspase-3, and caspase-9 activation. Further compounds were docked with Mcl-1 to confirm the mechanism of cytotoxicity. Compounds were confirmed by spectral techniques and screened for cytotoxicity at National Cancer Institute (USA). The active derivatives were screened by SRB and MTT. In addition, the potent compound was studied for apoptosis and cell cycle analysis by PI staining, Mcl-1 inhibition by TR-FRET assay, and activation assay of caspase-3 and caspase-9 with the Elisa technique. Compounds 6a and 6b exhibited the highest growth inhibition of 86.28% and 93.20% against SR and HOP- 62, respectively. Compound 6a showed higher cytotoxicity (IC50 = 0.4 μM) against THP-1 and HL-60. It showed 15- fold higher apoptosis compared to control by arresting cells at the Sub-G1 in the cell cycle. It also showed a potent inhibition with IC50 of 1.5 μM against the anti-apoptotic protein Mcl-1, which may induce apoptosis. Furthermore, apoptosis was evidenced by an increase in cleaved caspase-3 and caspase-9 to 4.20 and 3 folds, respectively higher than control. The docking score of compound 6a was in good agreement with the Mcl-1 inhibition assay. Compound 6a inhibited anti-apoptotic protein Mcl-1 and induced activation of pro-apoptotic proteins caspase-3 and caspase-9. These dual results suggested the mechanism of apoptosis and cytotoxicity.

Effective tumor cell abrogation via Venetoclax-mediated BCL-2 inhibition in KMT2A-rearranged acute B-lymphoblastic leukemia

Dysregulation of the intrinsic BCL-2 pathway-mediated apoptosis cascade is a common feature of hematological malignancies including acute B-lymphoblastic leukemia (B-ALL). The KMT2A-rearranged high-risk cytogenetic subtype is characterized by high expression of antiapoptotic protein BCL-2, likely due to the direct activating binding of KMT2A fusion proteins to the BCL2 gene. The BCL-2 inhibitor venetoclax (VEN) has proven great clinical value in other blood cancers, however, data on B-ALL is sparse and past studies have not so far described the effects of VEN on gene and protein expression profiles. Using cell lines and patient-derived in vivo xenograft models, we show BCL-2 pathway-mediated apoptosis induction and decelerated tumor cell counts in KMT2A-rearranged B-ALL but not in other cytogenetic subtypes. VEN treatment of cell line- and patient-derived xenografts reduced blast frequencies in blood, bone marrow, and spleen, and tumor cell doubling times were increased. Growth rates are further correlated with VEN concentrations in blood. In vitro incubation with VEN resulted in BCL-2 dephosphorylation and targeted panel RNA sequencing revealed reduced gene expression of antiapoptotic pathway members BCL2, MCL1, and BCL2L1 (BCL-XL). Reinforced translocation of BAX proteins towards mitochondria induced caspase activation and cell death commitment. Prolonged VEN application led to upregulation of antiapoptotic proteins BCL-2, MCL-1, and BCL-XL. Interestingly, the extrinsic apoptosis pathway was strongly modulated in SEM cells in response to VEN. Gene expression of members of the tumor necrosis factor signaling cascade was increased, resulting in canonical NF-kB signaling. This possibly suggests a previously undescribed mechanism of BCL-2-independent and NF-kB-mediated upregulation of MCL-1 and BCL-XL. In summary, we herein prove that VEN is a potent option to suppress tumor cells in KMT2A-rearranged B-ALL in vitro and in vivo. Possible evasion mechanisms, however, must be considered in subsequent studies.

S144: IMMUNE RESTORATION AND SYNERGISTIC ACTIVITY WITH FIRST-LINE (1L) IBRUTINIB (IBR) PLUS VENETOCLAX (VEN): TRANSLATIONAL ANALYSES OF CAPTIVATE PATIENTS WITH CLL

Background: Ibr and Ven work synergistically through their distinct and complementary modes of action: BTK inhibition by Ibr decreases levels of anti-apoptotic MCL-1 and BCL-XL proteins, but not BCL-2, and therefore increases sensitization to BCL-2 inhibition by Ven in vivo. Single agent Ibr has been shown to normalize CLL-associated immune cell alterations in number and function but the impact of the Ibr plus Ven combination (I+V) on immune cells has not been evaluated. Aims: To 1) confirm BCL-2 sensitization by single-agent Ibr and 2) monitor changes in the cellular immune profile of patients (pts) treated with I+V in CAPTIVATE (NCT02910583), a multicenter phase 2 study evaluating 1L I+V treatment (tx) in CLL and SLL. Methods: Ibr (420mg po daily) effects on anti-apoptotic proteins were evaluated by flow cytometry with samples from 4 previously untreated pts with CLL treated for 1 cycle (28 days). Immune restoration was evaluated in 79 previously untreated pts with CLL enrolled in the CAPTIVATE Minimal Residual Disease (MRD) cohort. Pts received 3 cycles of Ibr, followed by 12 cycles of I+V (Ibr 420 mg/d orally; Ven ramp-up to 400 mg/d orally). Pts who met criteria for confirmed undetectable MRD (Confirmed uMRD) were then randomized 1:1 to placebo fixed-duration (FD) tx or Ibr at cycle 16; pts who did not meet Confirmed uMRD criteria (uMRD Not Confirmed) were randomized 1:1 to Ibr or I+V. Cryopreserved peripheral blood mononuclear cells at baseline (pre-dose cycle 1) and day 1 of cycles 4, 7, 16, 20, 23, and 29 in each of the 4 arms were analyzed by high-dimensional flow cytometry (34-color panel). Immune cell subset counts of B cells, T cells, monocytes, dendritic cells (DCs), myeloid-derived suppressor cells, natural killer cells, and innate lymphoid cells were calculated and compared to those of 20 age-matched healthy donors. Median changes from baseline are reported. Results: In pts treated with single-agent Ibr for 1 cycle, MCL-1, BCL-XL and BCL-2 expression decreased by 74%, 95% and 10%, respectively, in lymph node emigrant (CD5+ CXCR4dim) CLL cells, confirming increased BCL-2-dependence and corresponding inhibitor sensitization. In CAPTIVATE pts treated with I+V, a rapid and significant decrease in circulating CLL cells was observed within the first 3 cycles of Ven tx initiation (Fig 1A, cycle 7). From cycle 16 and onwards, Confirmed uMRD pts had CLL cell counts similar to healthy donors (≤0.8 CLL cell/mL), while in uMRD Not Confirmed pts counts were 1.5 to 22.9 CLL cell/mL. Subsequently, normal B-cell count restoration occurred in Confirmed uMRD pts randomized to placebo (FD tx) with a recovery to levels similar to healthy donors (+332% at cycle 29; Fig 1B). Across all arms, abnormal counts of T cell subsets, classical monocytes and conventional DCs characteristic of CLL were normalized to healthy donor levels (‒49%, +101% and +91% respectively) within the first 6 months of tx and were maintained thereafter regardless of randomized tx. Plasmacytoid DCs counts recovered by cycle 20 (+598%) in all 4 arms. Image:Summary/Conclusion: Sensitization to BCL-2 inhibitor effects by Ibr supports the synergistic activity of I+V. The FD regimen (Confirmed uMRD, placebo arm) of I+V effectively eradicated CLL cells to healthy donor levels and enabled sustained regeneration of normal B cell counts in contrast to ongoing therapy in the remaining tx arms. Combination I+V also allowed for normalization of other critical immune cells, including T cell subsets, classical monocytes, and conventional and plasmacytoid DCs. These data demonstrate promising evidence of immune restoration with FD I+V tx.

Abstract 3338: Concurrent treatment with Pim kinase inhibitor enhances efficacy of FLT3 inhibitors in AML with FLT3-ITD through GSK-3βactivation and GSK-3β-mediated enhanced proteasomal degradation of c-Myc and Mcl-1

Abstract BACKGROUND: fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in acute myeloid leukemia (AML) cells in 30% of patients, with poor patient outcomes. FLT3 inhibitors are in clinical use, but with incomplete responses and onset of resistance. New targeting strategies are needed. The oncogenic serine/threonine kinase proviral integration site for Moloney murine leukemia virus (Pim-1) is upregulated downstream of FLT3-ITD, directly stimulates cell growth and inhibits apoptosis, and also phosphorylates and stabilizes FLT3 in a positive feedback loop. We previously showed that concurrent treatment of FLT3-ITD cells with Pim kinase and FLT3 inhibitors enhanced apoptosis induction through post-translational downregulation of the master transcription factor c-Myc and the anti-apoptotic protein Mcl-1. The serine/threonine kinase GSK-3β regulates c-Myc and Mcl-1 protein via phosphorylation at T58 and S159, respectively; we hypothesized downregulation of both proteins by combination treatment through phosphorylation by GSK-3β. METHODS: FLT3-ITD cells lines (Ba/F3-ITD, MV4-11, and MOLM-14) and primary FLT3-ITD AML patient blasts were cultured with the pan-Pim kinase inhibitor AZD1208, the FLT3 inhibitors gilteritinib or quizartinib and the GSK-3β inhibitor TCG-24. Protein expression was measured by immunoblotting. To measure protein half-lives, cells were pretreated with cycloheximide with and without the proteasome inhibitor MG-132. Ba/F3-ITD cells were infected with Myc-T58A plasmid, preventing c-Myc T58 phosphorylation, or Mcl-1 S159A plasmid, preventing Mcl-1 S159 phosphorylation, or empty vector controls. Apoptosis was measured by Annexin V labeling, measured by flow cytometry. RESULTS: Combined treatment of FLT3-ITD-expressing cell lines and AML patient blasts with Pim and FLT3 inhibitors caused rapid downregulation of c-Myc, and then Mcl-1, protein expression, relative to single drugs. Combined Pim and FLT3 inhibitor treatment caused marked decrease in half-lives of both proteins, which was rescued by proteasome inhibition, as the mechanism of protein downregulation. Combined Pim and FLT3 inhibitor treatment rapidly activated (dephosphorylated) GSK-3β and culture with GSK-3β inhibitor prevented induction of c-Myc and Mcl-1 downregulation, increased c-Myc and Mcl-1 protein turnover by concurrent Pim and FLT3 inhibitor treatment. Finally, combination treatment of Ba/F3-ITD cells infected with T58A c-Myc or S159A Mcl-1 plasmids did not downregulate these proteins or increase their turnover, with reduced apoptosis induction. CONCLUSION: Pim inhibitors enhance efficacy of FLT3 inhibitors via GSK-3β activation and GSK-3β-mediated enhanced proteasomal degradation of c-Myc and Mcl-1, highlighting GSK-3β as a key target in cells with FLT3-ITD. Citation Format: Jonelle Lee, Mario Scarpa, Aditi Chatterjee, Mooath Mustafa Ali, Prerna Singh, Shivani Kapoor, Rossana Trotta, Maria Baer. Concurrent treatment with Pim kinase inhibitor enhances efficacy of FLT3 inhibitors in AML with FLT3-ITD through GSK-3βactivation and GSK-3β-mediated enhanced proteasomal degradation of c-Myc and Mcl-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3338.

Abstract 5321: BRG399: A novel potent small molecule modulator of UBE2K for the treatment of various cancers

Abstract The ubiquitin-proteasome system (UPS) plays an important role in cellular processes for protein quality control and homeostasis and indeed, dysregulation within the pathway has implications in numerous diseases, especially cancer. Here, we introduce BRG399, a novel modulator of UBE2K - a unique E2 conjugating enzyme of the UPS, that we previously identified as a potential therapeutic target using BERG’s Interrogative Biology® platform. The anticancer potential of UBE2K modulation was validated by gene manipulation using siRNA mediated knockdown of UBE2K in several cancer cell models. Knockdown of UBE2K resulted in a 50% decrease in cell number in MIA PaCa2 cells and a 30% decrease in cell number in SKHEP1 and HepG2 cells at 96h post transfection. This effect was the result of a robust G2/M cell cycle arrest associated with increased CyclinB1 expression. In addition, a modest increase in apoptosis/necrosis (6-8%) was observed in cells with UBE2K knockdown. Using Fragment-Based Ligand Discovery and structure-based drug design, BRG399 was developed as a new chemical entity (NCE) which binds to UBE2K, resulting in modulation of several established canonical functions and an anti-cancer phenotype. In a panel of 102 cancer cell lines, BRG399 exhibited an anti-cancer activity in all cell lines tested as evident by extrapolation of IC50 and %Max Effect values from the dose response curves. In all but one cell line, double to triple digit nanomolar potency was observed ranging from 17nM to 318 nM and viability of 76 cell lines was reduced by more than 70%. The anti-cancer potency of BRG399 was also established in a multi-tumor type panel of patient-derived organoids (PDO) of which 68% were highly or partially responsive to BRG399 as indicated by 37-73% decrease in PDO viability. Mechanistic studies demonstrated that BRG399’s anti-cancer effect was a result of a robust G2/M cell cycle arrest and apoptosis mediated cell death. Molecular events associated with G2/M arrest included stabilization of CyclinB1 and sustained activation of cyclin-dependent kinase CDK1, while those associated with apoptosis involved decreased expression of anti-apoptotic proteins Bcl-XL and Mcl-1 and increased expression of cleaved PARP (cPARP) and Capase 3. Together, these results support a novel role for UBE2K in the regulation of the cell cycle in cancer cells and demonstrate that its modulation by BRG399 influences phenotypic end-points consistent with an anti-cancer effect. Citation Format: Maria-Dorothea Nastke, Marisa Permatteo, Pragalath Sundararajan, Shefali Sharma, Jacob Matson, Kaleigh Gray, Arcan Guven, Shiva Kazerounian, Anne Diers, Rangaprasad Sarangarajan, Niven R. Narain, Vivek K. Vishnudas, Stephane Gesta. BRG399: A novel potent small molecule modulator of UBE2K for the treatment of various cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5321.

Abstract 5652: A novel anti apoptotic function of CTLA4 in melanoma

Abstract Cytotoxic T Lymphocyte Antigen 4 (CTLA4) is an important immune checkpoint protein and has been utilized as an immunotherapeutic target against melanoma. Our previous studies have shown that CTLA4 is expressed at low levels in primary human melanocytes but is upregulated in mutant-BRAF/NRAS melanoma cells and tissues. Despite predominantly intracellular localization of CTLA4, its intracellular function remains highly contradictory and unsubstantiated. We found that ectopic expression of Ctla4 in mouse melanoma cell lines substantially promoted lung colonization in allograft model systems in syngeneic mice. Moreover, similar results were observed in immunocompromised recipient mice, suggesting an intracellular cell-autonomous tumorigenic role of Ctla4 in melanoma. These finding led us to investigated intracellular functions of CTLA4 and its potential roles in melanomagenesis. Through the Ingenuity Pathway Analysis of our proteomics data, we found apoptosis as a major affected pathway in Ctla4-expressing mouse melanoma cells. We treated the cells (B2905-Ctla4-ee and EV control) with doxorubicin to induce apoptosis, followed by assessment by Annexin V assay. We found that Ctla4 expressing mouse melanoma cells were significantly resistant to doxorubicin-induced apoptosis. In reciprocal experiment, we generated (by CRISPR-Cas9) CTLA4-knockout A2058 human melanoma cells that exhibit high endogenous CTLA4 expression and we found that knockout cells showed significantly increased apoptosis than the parental cells. Moreover, we also observed significant upregulation of anti-apoptotic proteins (Bnip3, Birc6, Pak1, Mcl-1, Survivin, Rac1/Cdc42, Bcl-2, and Bnip3l) and downregulation of pro-apoptotic proteins (p53 and Bad) in CTLA4-expressing human melanoma cell lines. CTLA4-expressing cells also showed significantly higher invasion in Matrigel-coated transwell assay. These findings lead us to conclude that CTLA4 plays a significant role in regulating apoptosis and promoting melanoma progression in BRAF/NRAS mutant cells. Citation Format: Hasan Raza Kazmi, Xuan Mo, Bo Zhou, Sara Preston-Alp, Scott Gross, Hassaan Wajeeh, Carmen Merali, Jonathan Soboloff, Salim Merali, M. Raza Zaidi. A novel anti apoptotic function of CTLA4 in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5652.

Abstract 1793: Combination treatment with a MCL1 Inhibitor AZD5991 overcomes resistance to MEK inhibitor in TNBC cells

Abstract Background: Triple-negative breast cancer (TNBC), which is associated with poor prognosis, accounts for 15-20% of breast cancers (BCs) but up to 30% of BC deaths, likely because of its high recurrence rate and limited targeted therapy options. Basal type-BC cells are sensitive to MEKi (MEK Inhibitor), and 77% of basal-type BCs are TNBCs. We previously showed that MEKi selumetinib (AZD6244) effectively inhibits tumor growth and metastasis in a TNBC xenograft model. However, clinical studies have shown that MEKi as single agents have only modest activity against solid tumors. Therefore, combinatorial therapeutic strategies represent an urgent unmet clinical need for TNBC patients. Using a high-throughput whole-genome siRNA screen, we identified and validated the antiapoptotic protein MCL1, which is overexpressed in many human cancers, including TNBC, as a potential candidate that could be therapeutically targeted alongside MEK. We hypothesized that the combination of AZD6244 plus AZD5991 overcomes MEKi resistance in TNBC. Methods and Results: We previously established two cell lines resistant to AZD6244 to a final concentration of 15 µM (MDA-MB-231-R and SUM-149-R). Both these MDA MB-231-R and SUM-149-R cells showed increased proliferation with IC50 of 24.01 and 22.76μM as compared to the parental IC50 2.7 and 3.80µM respectively. The colony-forming rates of the MDA-MB-231-R and SUM-149-R cells were more than 50% higher than their parental cells (p ≤ 0.05), and the resistant cells had lower proportions of the cells in G0/G1 phase (49.03% and 22.89%, respectively) than their parental cells did (73.01% and 60.03%, respectively). Compared with their parental cells, SUM 149-R (86.26% vs 66.04%) and MDA MB-231-R (95.27% vs. 73.47%) had a higher proportion of CD44+CD24-/low cells and higher average mammosphere count (330 vs. 55) and (892 vs. 273). The IC50 values for SUM149-R (1.68 µM) and MDA-MB-231-R (7.2 µM) cells treated with AZD6244 in combination with the MCL inhibitor AZD5991 was significantly lower than that for cells treated with AZD6244 alone. To validate this finding, we used siRNA to knock down MCL1 and confirmed 80-90% silencing of both resistant and parental cells via western blotting. Furthermore, MCL1 silencing sensitized both MDA-MB-231-R and SUM-149-R cells to AZD6244, as MCL1-knockout cells treated with AZD6244 had significant reduction in cell proliferation and colony formation as compared to the respective controls. Conclusions and Future Directions: Our findings support our hypothesis that the combination of AZD6244 plus AZD5991 overcomes MEKi resistance in TNBC. To further translate these interesting findings, we plan to evaluate the effect of this drug combination in vivo in TNBC models. We have also performed a whole-proteome analysis of MDA-MB-231 parental and MDA-MB-231-R cell lines using LC-MS/MS and are now validating the genes/pathways involved in MEK resistance Citation Format: Mohd Mughees, Moises Tacam, Alex Tan, Lakesla Iles, Michael White, Debu Tripathy, Geoffrey Bartholomeusz, Chandra Bartholomeusz. Combination treatment with a MCL1 Inhibitor AZD5991 overcomes resistance to MEK inhibitor in TNBC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1793.

Abstract 363: Intracellular redox milieu regulates Mcl-1 and decreases overall mitochondrial priming in hematopoietic cancers

Abstract Background: The dichotomy of redox signaling in cancer cell fate decisions has emerged as a topic of importance over the years. In particular, our group has highlighted the role of superoxide (O2 .-)-mediated signaling in the acquisition of drug resistance and cancer survival. Mechanistically, this involves post-translational modification of proteins involved in oncogenic signaling, such as c-Myc and NF-κB, as well as apoptosis resistance like Bcl-2 at the serine 70 phosphorylation site. Here we present evidence that O2 .--mediated signaling stabilizes anti-apoptotic protein Mcl-1, a commonly amplified gene in human cancers and is implicated in resistance to venetoclax, the Bcl-2 specific BH3 mimetic. Methods: Intracellular O2 .- levels was increased either pharmacologically or by siRNA mediated knockdown of SOD1 in MOLM14, RPMI8226, OCI-AML3 hematopoietic cell lines. Cell viability and apoptosis were assessed by MTT and Annexin V/DAPI staining. Protein expression was determined by Western blot analysis. BH3 profiling of patient-derived CLL cells was used to assess mitochondrial priming. Results: Elevated O2 .- levels increased Mcl-1 stability through Thr163 phosphorylation of Mcl-1, which in turn reduced ubiquitination-dependent degradation. Furthemore, venetoclax resistant cells possessed higher O2 .- levels and increase in intracellular O2 .- levels promoted resistance to Mcl-1 inhibitor, S63845 through increased binding between Mcl-1 and pro-apoptotic proteins NOXA and Bak. Contrarily, scavenging of O2 .- re-sensitized venetoclax-resistant leukemia cells to venetoclax. Importantly, BH3 profiling revealed that increase in intracellular O2 .- levels decreased overall mitochondrial priming in primary CLL cells by promoting sequestration of pro-apoptotic proteins that specifically target Mcl-1. Conclusion: Oncogenic activity of O2 .- is mediated by increased Mcl-1 stability through Thr163 phosphorylation, which resulted in a reduction in overall mitochondrial priming and increased binding of Mcl-1 to NOXA and Bak. Scavenging O2 .- reduces Mcl-1 levels and sensitizes resistant cells to venetoclax-induced apoptosis thereby demonstrating that manipulation of the redox microenvironment could be a potential vulnerability to be exploited in venetoclax resistant cancers Citation Format: Jolin Xiao Lai, Stephen Jun Chong, Matthew Davids, Shazib Pervaiz. Intracellular redox milieu regulates Mcl-1 and decreases overall mitochondrial priming in hematopoietic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 363.

Abstract SY05-02: Applications of base editor technology in small molecule: Target validation

Abstract CRISPR screens that focus on the gene as the unit of information - whether using knockout, interference, or activation - have reinvigorated the field of functional genomics. The recent development of Base Editor technology allows nucleotide-level manipulation, enabling fine-scale investigation of endogenous loci. The low upfront costs of generating a pooled base editing library, coupled with the small scale and relative ease of executing such screens, makes the financial barrier to entry for such experiments low while also not requiring deep experience with screening. One application of this technology is screens that aim to identify resistance mutations in putative targets of small molecules, which provides excellent evidence that the phenotype is caused by on-target activity of the small molecule. This has heretofore typically been an extraordinarily laborious and time-consuming experimental path, by either slowly cultivating spontaneous drug resistant mutants or by generating and screening large scale ORF-variant pools. Base editor screens are well-suited to vastly accelerate this process. Using both A>G and C>T base editors, coupled with variants of the SpCas9 enzyme, we have conducted drug resistance screens with small molecules that target the anti-apoptotic proteins MCL1, BCL2L1, and BCL2, as well as the DNA repair regulator PARP1. These screens identified resistance residues that were also observed in clinical samples, illustrating the utility of this approach. Additionally, base editor screens are excellent tools for studying genetic variants, as we will demonstrate with screens identifying loss-of-function mutations in BRCA1, BRCA2, and TP53. We anticipate that the tools and methodologies described here will facilitate the investigation of impactful variants at a finer and deeper resolution for any locus of interest. Citation Format: Audrey Griffith, Annabel Sangree, Priyanka Roy, Ruth Hanna, John Doench. Applications of base editor technology in small molecule: Target validation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY05-02.

Biphasic Dose-Dependent G0/G1 and G2/M Cell-Cycle Arrest by Synthetic 2,3-Arylpyridylindole Derivatives in A549 Lung Cancer Cells.

A collection of 2,3-arylpyridylindole derivatives were synthesized via the Larock heteroannulation and evaluated for their in vitro cytotoxic activity against A549 human lung cancer cells. Two derivatives expressed good cytotoxicity with IC50 values of 1.18±0.25 μM and 0.87±0.10 μM and inhibited tubulin polymerization in vitro, with molecular docking studies suggesting the binding modes of the compounds in the colchicine binding site. Both derivatives have biphasic cell cycle arrest effects depending on their concentrations. At a lower concentration (0.5 μM), the two compounds induced G0/G1 cell cycle arrest by activating the JNK/p53/p21 pathway. At a higher concentration (2.0 μM), the two derivatives arrested the cell cycle at the G2/M phase via Akt signaling and inhibition of tubulin polymerization. Additional cytotoxic mechanisms of the two compounds involved the decreased expression of Bcl-2 and Mcl-1 antiapoptotic proteins through inhibition of the STAT3 and Akt signaling pathways.

Aryl Hydrocarbon Receptor Promotes Cell Growth, Stemness Like Characteristics, and Metastasis in Human Ovarian Cancer via Activation of PI3K/Akt, β-Catenin, and Epithelial to Mesenchymal Transition Pathways.

Ovarian cancer (OC) ranks first in cancer-related deaths out of all female reproductive malignancies with high-pitched tumor relapse and chemoresistance. Several reports correlate cancer occurrences with exposure to xenobiotics via induction of a protein receptor named aryl hydrocarbon receptor (AhR). However, the effect of AhR on OC proliferation, expansion, and chemoresistance remains unrevealed. For this purpose, OC cells A2780 and A2780cis cells were treated with AhR activator, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and the effects were determined by Real-Time Cell Analyzer, clonogenic assay, flow cytometry, immunoblotting and wound healing assay. Our results showed that activation of AhR by TCDD in A2780 cells induced the PI3K/AKT pathway followed by induction of anti-apoptotic proteins BCL-2, BCL-xl, and MCL-1. In addition, a significant increase in stemness marker aldehyde dehydrogenase (ALDH1) was observed. This effect was also associated with an accumulation of β-catenin, a Wnt transcription factor. Moreover, we observed induction of epithelial to mesenchymal transition (EMT) upon AhR activation. In conclusion, the results from the current study confirm that AhR mediates OC progression, stemness characteristics, and metastatic potential via activation of PI3K/Akt, Wnt/β-catenin, and EMT. This study provides a better insight into the modulatory role of AhR that might help in developing novel therapeutic strategies for OC treatment.

Design, synthesis, biological evaluation, and in silico studies of 2-aminobenzothiazole derivatives as potent PI3Kα inhibitors.

A new series of 2-aminobenzothiazole derivatives was designed, synthesized and evaluated for their anticancer activity against the MCF7, MDAMB-231, and HepG2 cancer cell lines. All synthesized derivatives (8a-8n) demonstrated moderate to high anticancer activity against the tested cell lines. As the most potent compound in the series, compound 8i displayed excellent inhibitory potency with an IC50 value of 6.34 μM and compound 8m displayed an IC50 value of 8.30 µM against the MCF7 cell line compared to the standard drug HS-173 (IC50 = 10.25 μM). PI3K enzyme activity assays demonstrated that compound 8i is highly selective against PI3Kα, with an IC50 value of 1.03 nM. Wound healing assays and cell cycle analysis of compounds 8i and 8m revealed that both compounds suppressed the migration of MCF7 cells and induce cell cycle arrest in the S phase. In the cell death assay, compound 8i was revealed to induce apoptosis in a dose-dependent pattern; further Western blot assays revealed that compound 8i obviously decreases the levels of the antiapoptotic proteins Bcl-xL and Mcl-1. Downregulation of the expression of p-Akt confirmed the PI3K inhibitory activity of compound 8i. The molecular docking and molecular dynamicssimulation studies performed were found in agreement with the PI3Kα inhibitory activity assessments performed experimentally.

How Phosphofructokinase-1 Promotes PI3K and YAP/TAZ in Cancer: Therapeutic Perspectives.

Simple SummaryWe propose that PFK1 promotes a positive feedback loop with PI3K/AKT and YAP/TAZ signaling pathways in cancer cells. Therefore, targeting PFK1 (or its product F-1,6-BP) could improve the efficacy of PI3K and YAP/TAZ inhibitors currently tested in clinical trials. To this aim, we suggest the use of citrate, which is a physiologic and potent inhibitor of PFK1.PI3K/AKT is one of the most frequently altered signaling pathways in human cancers, supporting the activation of many proteins sustaining cell metabolism, proliferation, and aggressiveness. Another important pathway frequently altered in cancer cells is the one regulating the YAP/TAZ transcriptional coactivators, which promote the expression of genes sustaining aerobic glycolysis (such as WNT, MYC, HIF-1), EMT, and drug resistance. Of note, the PI3K/AKT pathway can also regulate the YAP/TAZ one. Unfortunately, although PI3K and YAP inhibitors are currently tested in highly resistant cancers (both solid and hematologic ones), several resistance mechanisms may arise. Resistance mechanisms to PI3K inhibitors may involve the stimulation of alternative pathways (such as RAS, HER, IGFR/AKT), the inactivation of PTEN (the physiologic inhibitor of PI3K), and the expression of anti-apoptotic Bcl-xL and MCL1 proteins. Therefore, it is important to improve current therapeutic strategies to overcome these limitations. Here, we want to highlight how the glycolytic enzyme PFK1 (and its product F-1,6-BP) promotes the activation of both PI3K/AKT and YAP/TAZ pathways by several direct and indirect mechanisms. In turn, PI3K/AKT and YAP/TAZ can promote PFK1 activity and F-1,6-BP production in a positive feedback loop, thus sustaining the Warburg effect and drug resistance. Thus, we propose that the inhibition of PFK1 (and of its key activator PFK2/PFKFB3) could potentiate the sensitivity to PI3K and YAP inhibitors currently tested. Awaiting the development of non-toxic inhibitors of these enzymes, we propose to test the administration of citrate at a high dosage, because citrate is a physiologic inhibitor of both PFK1 and PFK2/PFKFB3. Consistently, in various cultured cancer cells (including melanoma, sarcoma, hematologic, and epithelial cancer cells), this “citrate strategy” efficiently inhibits the IGFR1/AKT pathway, promotes PTEN activity, reduces Bcl-xL and MCL1 expression, and increases sensitivity to standard chemotherapy. It also inhibits the development of sarcoma, pancreatic, mammary HER+ and lung RAS-driven tumors in mice without apparent toxicities.

Methylation of p53-responsive oncosuppressive microRNA genes in hemoblastosis

The purpose of the study was to present up-to-date data on the frequency and significance of a number of p53-responsive oncosuppressive micrornas genes methylation in malignant neoplasms of the blood system.Material and methods. The search for available literary sources published in the Pubmed and RISC databases was carried out. A total of 399 articles were found, of which 62 were included in this review.Results. The p53 protein regulates a whole class of microRNAs – highly conserved small RNA molecules that affect gene expression mainly by suppressing translation. МicroRNAs play an important role in all cellular processes and can have both oncosuppressive and pro-oncogenic properties. Impaired expression of p53-activated oncosuppressive micrornas in various tumors may be associated with specific epigenetic mechanisms (DNA methylation and histone deacetylation). The review examines the molecular and genetic characteristics of oncosuppressive micrornas functioning in normal hematopoiesis, the violation of expression of which is shown in the development of hemoblastoses, namely: miR-34a, miR-34b/c, miR-145, miR-143 and miR-203. It is known that the transcription of the genes of these microRNAs is carried out and regulated from their own promoters. The latest published research results on the diagnostic, prognostic and clinical significance of gene methylation of the microRNAs under consideration in malignant neoplasms of the blood system are presented. According to literature data, common targets for mir-34a, mir-34b/c, mir-145, mir-143 and miR-203 microRNAs are mRNAs of a number of pro-oncogenes, namely: transcription factor C-MYC, positive cell cycle regulators at the G1/S transition point of CDK4, CDK6 and CYCLIN-D1 phases, anti-apoptotic proteins MDM2, MDM4, BCL2 and MCL1, as well as DNMT3A and DNMT3B methyltransferases and other molecules. In this regard, it should be noted that there are positive feedbacks between p53 and microRNAs activated by it, as well as negative feedbacks between p53-responsive micrornas and C-MYC and DNA methyltransferases.Conclusion. Thus, the data presented in the review clarify the current understanding of the work of the regulatory network of the p53 protein and the micrornas activated by it, and also emphasize the functional association of p53-responsive microRNAs.

Aryl hydrocarbon Receptor (AhR) Promotes Cell Growth, Induces Stemness Like Characteristics and Metastasis in Ovarian Cancer Cells via Activation of Akt, β‐Catenin and EMT

BackgroundOvarian cancer is the most lethal form of all gynecological malignancies with highpitched tumor relapse and chemoresistance. Cancer stem cells (CSCs), a small sub population of cancer cells driving tumor initiation, progression, and metastasis, are known to be a major contributor of therapy resistance. Recent studies have depicted a link between CSC development and exposure to environmental pollutants like 2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin (TCDD). Reports suggest that one of the major pathways that mediate the metabolism of these environmental chemicals is the aryl hydrocarbon receptor (AhR) pathway, whose role in several CSCs is lately demonstrated. However, its role in ovarian cancer development and chemoresistance remains uninvestigated. We hypothesize that AhR and its regulatory genes CYP1A1 and CYP1B1 are involved in the ovarian cancer progression and chemoresistance. The objectives of the present study were a) to examine the role and involvement of AhR in ovarian cancer progression and chemoresistance and b) to explore the underlying mechanisms.MethodsTo test our hypothesis, seven ovarian cancer cell lines; A2780, A2780cis (cisplatin resistant), OVCAR‐3, SKOV‐3, CAOV‐3, and SW‐626 were utilized in this study. Ovarian cancer cells were treated for 24 h with a potent AhR activator, tetrachlorodibenzo‐p‐dioxin (TCDD, 10 nM), thereafter protein expression of AhR, CYP1A1, CYP1B1, pro‐ and anti‐apoptotic, and stemness markers were determined by immunoblotting.Resultsour study showed that SKOV‐3 cells showed highest basal expression of AhR, whereas A2780cis and A2780 expressed the highest basal expression of CYP1A1 and CYP1B1 proteins, respectively. At the inducible levels, treatment of selected cells (OVCAR‐3, SKOV‐3 and A2780) with TCDD significantly induced the protein level of AhR, CYP1A1 in OVCAR‐3 and SKOV‐3, and CYP1B1 in only SKOV‐3 and A2780 cells. To investigate the mechanism involved, we utilized A2780 cells as a model to explore the effect of TCDD on pro‐ and anti‐apototic, Epithelial to Mesenchymal Transition (EMT), and stemness markers. Our results showed that activation of AhR by TCDD in A2780 cells induced phosphorylation of Akt protein with a concomitant increase in the expression of anti‐apoptotic proteins BCL‐2, BCL‐xl and MCL‐1. In addition, a significant increase of aldehyde dehydrogenase (ALDH1), a functional CSC‐specific marker exhibiting important role in cancer progression and chemoresistance was also observed. This effect was also associated with an accumulation of βCatenin, a Wnt transcription factor. Moreover, we found a decrease in E‐cadherin, an epithelial marker, followed by a concurrent increase in Vimentin, a mesenchymal marker, upon AhR activation, confirming the induction of EMT.ConclusionThe results from our study indicate that AhR regulates ovarian cancer progression, exerts stemness characteristics and induces metastasis via activation of Akt, Wnt/β‐Catenin and EMT. This study provides a better understanding of the regulation of ovarian cancer cells in relation to AhR that could help in devising novel therapeutics for better management of ovarian cancer and prevention.

Involvement of Akt and the antiapoptotic protein Bcl-xL in the inhibition of apoptosis of dendritic cells by Leishmania mexicana.

The intracellular parasite Leishmania mexicana inhibits camptothecin (CPT)-induced apoptosis of monocyte-derived dendritic cells (moDC) through the down-regulation of p38 and JNK phosphorylation, while the kinase Akt is maintained active for 24 h. In addition, the infection of moDC with L. mexicana promastigotes increases the protein presence of the antiapoptotic protein Bcl-xL. In the present work, we aimed to investigate the role of Akt in the inhibition of apoptosis of moDC by L. mexicana and in the modulation of the expression of the antiapoptotic proteins Bcl-2, Mcl-1 and Bcl-xL. moDC were infected with L. mexicana metacyclic promastigotes and treated with CPT, an Akt inhibitor, or both and the mitochondrial outer membrane permeabilization (MOMP) and protein presence of active caspase 3, Bcl-2, Mcl-1 and Bcl-xL were evaluated. Our results show that the specific inhibition of Akt reverts the apoptosis protective effect exerted by L. mexicana on moDC reflected by a reduction in MOMP, caspase 3 activation, and upregulation of Bcl-xL. Interestingly, we also found that the infection of moDC with L. mexicana promastigotes induces a decrease in Bcl-2 along with an isoform change of Mcl-1, this independently to Akt activity. We demonstrated that Akt is deeply involved in the inhibition of apoptosis of moDC by L. mexicana.

Cyclin-dependent kinase inhibitor fadraciclib (CYC065) depletes anti-apoptotic protein and synergizes with venetoclax in primary chronic lymphocytic leukemia cells

Fadraciclib (CYC065) is a second-generation aminopurine CDK2/9 inhibitor with increased potency and selectivity toward CDK2 and CDK9 compared to seliciclib (R-roscovitine). In chronic lymphocytic leukemia (CLL), a disease that depends on the over-expression of anti-apoptotic proteins for its survival, inhibition of CDK9 by fadraciclib reduced phosphorylation of the C-terminal domain of RNA polymerase II and blocked transcription in vitro; these actions depleted the intrinsically short-lived anti-apoptotic protein Mcl-1 and induced apoptosis. While the simulated bone marrow and lymph node microenvironments induced Mcl-1 expression and protected CLL cells from apoptosis, these conditions did not prolong the turnover rate of Mcl-1, and fadraciclib efficiently abrogated the protective effect. Further, fadraciclib was synergistic with the Bcl-2 antagonist venetoclax, inducing more profound CLL cell death, especially in samples with 17p deletion. While fadraciclib, venetoclax, and the combination each had distinct kinetics of cell death induction, their activities were reversible, as no additional cell death was induced upon removal of the drugs. The best combination effects were achieved when both drugs were maintained together. Altogether, this study provides a rationale for the clinical development of fadraciclib in CLL, either alone or in combination with a Bcl-2 antagonist.

Dual BTK/SYK inhibition with CG-806 (luxeptinib) disrupts B-cell receptor and Bcl-2 signaling networks in mantle cell lymphoma

Aberrant B-cell receptor (BCR) signaling is a key driver in lymphoid malignancies. Bruton tyrosine kinase (BTK) inhibitors that disrupt BCR signaling have received regulatory approvals in therapy of mantle cell lymphoma (MCL). However, responses are incomplete and patients who experience BTK inhibitor therapy failure have dire outcomes. CG-806 (luxeptinib) is a dual BTK/SYK inhibitor in clinical development in hematologic malignancies. Here we investigated the pre-clinical activity of CG-806 in MCL. In vitro treatment with CG-806 thwarted survival of MCL cell lines and patient-derived MCL cells in a dose-dependent manner. CG-806 blocked BTK and SYK activation and abrogated BCR signaling. Contrary to ibrutinib, CG-806 downmodulated the anti-apoptotic proteins Mcl-1 and Bcl-xL, abrogated survival of ibrutinib-resistant MCL cell lines, and partially reversed the pro-survival effects of stromal microenvironment-mimicking conditions in primary MCL cells. Dual BTK/SYK inhibition led to mitochondrial membrane depolarization accompanied by mitophagy and metabolic reprogramming toward glycolysis. In vivo studies of CG-806 demonstrated improved survival in one of the two tested aggressive MCL PDX models. While suppression of the anti-apoptotic Bcl-2 family proteins and NFκB signaling correlated with in vivo drug sensitivity, OxPhos and MYC transcriptional programs were upregulated in the resistant model following treatment with CG-806. BAX and NFKBIA were implicated in susceptibility to CG-806 in a whole-genome CRISPR-Cas9 library screen (in a diffuse large B-cell lymphoma cell line). A high-throughput in vitro functional drug screen demonstrated synergy between CG-806 and Bcl-2 inhibitors. In sum, dual BTK/SYK inhibitor CG-806 disrupts BCR signaling and induces metabolic reprogramming and apoptosis in MCL. The Bcl-2 network is a key mediator of sensitivity to CG-806 and combined targeting of Bcl-2 demonstrates synergy with CG-806 warranting continued exploration in lymphoid malignancies.