Synergistic Induction Of Apoptosis Research Articles

Venetoclax resistance in acute lymphoblastic leukemia is characterized by increased mitochondrial activity and can be overcome by co-targeting oxidative phosphorylation

Deregulated apoptosis signaling is characteristic for many cancers and contributes to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Apoptosis is controlled by different pro- and anti-apoptotic molecules. Inhibition of anti-apoptotic molecules like B-cell lymphoma 2 (BCL-2) has been developed as therapeutic strategy. Venetoclax (VEN), a selective BCL-2 inhibitor has shown clinical activity in different lymphoid malignancies and is currently evaluated in first clinical trials in BCP-ALL. However, insensitivity to VEN has been described constituting a major clinical concern. Here, we addressed and modeled VEN-resistance in BCP-ALL, investigated the underlying mechanisms in cell lines and patient-derived xenograft (PDX) samples and identified potential strategies to overcome VEN-insensitivity. Leukemia lines with VEN-specific resistance were generated in vitro and further characterized using RNA-seq analysis. Interestingly, gene sets annotated to the citric/tricarboxylic acid cycle and the respiratory electron transport chain were significantly enriched and upregulated, indicating increased mitochondrial metabolism in VEN-resistant ALL. Metabolic profiling showed sustained high mitochondrial metabolism in VEN-resistant lines as compared to control lines. Accordingly, primary PDX-ALL samples with intrinsic VEN-insensitivity showed higher oxygen consumption and ATP production rates, further highlighting that increased mitochondrial activity is a characteristic feature of VEN-resistant ALL. VEN-resistant PDX-ALL showed significant higher mitochondrial DNA content and differed in mitochondria morphology with significantly larger and elongated structures, further corroborating our finding of augmented mitochondrial metabolism upon VEN-resistance. Using Oligomycin, an inhibitor of the complex V/ATPase subunit, we found synergistic activity and apoptosis induction in VEN-resistant BCP-ALL cell lines and PDX samples, demonstrating that acquired and intrinsic VEN-insensitivity can be overcome by co-targeting BCL-2 and the OxPhos pathway. These findings of reprogrammed, high mitochondrial metabolism in VEN-resistance and synergistic activity upon co-targeting BCL-2 and oxidative phosphorylation strongly suggest further preclinical and potential clinical evaluation in VEN-resistant BCP-ALL.

Abstract LB451: Targeting oncogenic transcription and signaling crosstalk fueling drug resistance in lung cancer

Abstract Lung cancer is the leading cause of all cancer-related mortality worldwide. The RAS-RAF-MEK-ERK (RAS-MAPK) signaling pathway is critical for maintaining cell survival and proliferation. Somatic mutations in the RAS or RAF genes are associated with frequent hyper-activation of RAS-MAPK pathway in non-small cell lung cancer or NSCLC. While direct inhibitors of KRAS G12C are emerging with promising efficacy, resistance to these agents as well as to inhibitors of RAF and MEK remains an obstacle to long-term patient survival. Therefore, it is essential to understand how drug resistance emerges in lung cancer and to identify bypass survival pathways or compensatory mechanisms that limit the response to RAS-MAPK targeted therapies in lung cancer. Emerging literature indicates a critical role of bromodomain and extra-terminal domain (BET) family of bromodomain proteins (BRD4, BRD3, BRD2) in various human malignancies and provides the rational for targeting BET proteins as a strategy for the development of new anticancer drugs. Using combinatorial drug screens in a panel of NSCLC cell lines, we found that BET proteins act in parallel to the RAS-MAPK pathway to promote drug resistance in lung cancer. Combination of BET and RAS-MAPK inhibitors caused pronounced cell death and potent tumor regression/stasis in several KRAS or BRAF mutant models of NSCLC with synergistic apoptosis induction and downregulation of MYC, a transcriptional target of BET proteins. However, concurrent loss of the tumor suppressor STK11 (aka LKB1) in NSCLC cells induced activation of the Hippo pathway effector and transcriptional co-activator, YAP and conferred resistance to the BET and RAS-MAPK inhibitors combination in NSCLC cells and tumors.These findings suggest an unprecedented functional interplay between BET chromatin regulators, Hippo-YAP and RAS-MAPK signaling in lung cancer and therapy resistance. Further, this study identified STK11 as a modifier of sensitivity to the BET and RAS-MAPK inhibitors combination in NSCLC cells and tumors. Citation Format: Nilanjana Chatterjee, Victor Olivas, Wei Wu, Tracy Tang, Ben Powell, Trever Bivona. Targeting oncogenic transcription and signaling crosstalk fueling drug resistance in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB451.

Synergistic Effects of the RARalpha Agonist Tamibarotene and the Menin Inhibitor Revumenib in Acute Myeloid Leukemia Cells with KMT2A Rearrangement or NPM1 Mutation.

Inhibition of menin in acute myeloid leukemia (AML) harboring histone-lysine-N-methyltransferase 2A rearrangement (KMT2Ar) or the mutated Nucleophosmin gene (NPM1c) is considered a novel and effective treatment approach in these patients. However, rapid acquisition of resistance mutations can impair treatment success. In patients with elevated retinoic acid receptor alpha (RARA) expression levels, promising effects are demonstrated by the next-generation RARalpha agonist tamibarotene, which restores differentiation or induces apoptosis. In this study, the combination of revumenib and tamibarotene was investigated in various KMT2Ar or NPM1c AML cell lines and patient-derived blasts, focusing on the potential synergistic induction of differentiation or apoptosis. Both effects were analyzed by flow cytometry and validated by Western blot analysis. Synergy calculations were performed using viability assays. Regulation of the relevant key mediators for the MLL complex were quantified by RT-qPCR. In MV4:11 cells characterized by the highest relative mRNA levels of RARA, highly synergistic induction of apoptosis is demonstrated upon combination treatment. Induction of apoptosis by combined treatment of MV4:11 cells is accompanied by pronounced induction of the pro-apoptotic protein BAX and a synergistic reduction in CDK6 mRNA levels. In MOLM13 and OCI-AML3 cells, an increase in differentiation markers like PU.1 or a decreased ratio of phosphorylated to total CEBPA is demonstrated. In parts, corresponding effects were observed in patient-derived AML cells carrying either KMT2Ar or NPM1c. The impact of revumenib on KMT2Ar or NPM1c AML cells was significantly enhanced when combined with tamibarotene, demonstrating synergistic differentiation or apoptosis initiation. These findings propose promising strategies for relapsed/refractory AML patients with defined molecular characteristics.

Synergistic induction of apoptosis in lung cancer cells through co-delivery of PLGA phytol/α-bisabolol nanoparticles.

This study explored the potential of poly-(lactic-co-glycolic) acid (PLGA) nanoparticles to enhance the effectiveness of anticancer treatments through combination therapy with phytol and α-bisabolol. The encapsulation efficiency of the nanoparticles was investigated, highlighting the role of ionic interactions between the drugs and the polymer. Characterization of PLGA-Phy+Bis nanoparticles was carried out using DLS with zeta potential and HR-TEM for size determination. Spectrophotometric measurements evaluated the encapsulation efficiency, loading efficiency, and in vitro drug release. FTIR analysis assessed the chemical interactions between PLGA and the drug actives, ensuring nanoparticle stability. GC-MS was employed to analyze the chemical composition of drug-loaded PLGA nanocarriers. Cytotoxicity was evaluated via the MTT assay, while Annexin V-FITC/PI staining and western blot analysis confirmed apoptotic cell death. Additionally, toxicity tests were performed on L-132 cells and in vivo zebrafish embryos. The study demonstrates high encapsulation efficiency of PLGA-Phy+Bis nanoparticles, which exhibit monodispersity and sizes of 189.3±5nm (DLS) and 268±54 nm (HR-TEM). Spectrophotometric analysis confirmed efficient drug encapsulation and release control. FTIR analysis revealed nanoparticle structural stability without chemical interactions. MTT assay results demonstrated the promising anticancer potential of all thethree nanoparticle types (PLGA-Phy, PLGA-Bis, and PLGA-Phy+Bis) against lung cancer cells. Apoptosis was confirmed through Annexin V-FITC/PI staining and western blot analysis, which also revealed changes in Bax and Bcl-2 protein expression. Furthermore, the nanoparticles exhibited non-toxicity in L-132 cells and zebrafish embryo toxicity tests. PLGA-Phy+Bis nanoparticles exhibited efficient encapsulation, controlled release, and low toxicity. Apoptosis induction in A549 cells and non-toxicity in healthy cells highlight their clinical potential.

Enhanced TP53 reactivation disrupts MYC transcriptional program and overcomes venetoclax resistance in acute myeloid leukemias.

The tumor suppressor TP53 is frequently inactivated in a mutation-independent manner in cancers and is reactivated by inhibiting its negative regulators. We here cotarget MDM2 and the nuclear exporter XPO1 to maximize transcriptional activity of p53. MDM2/XPO1 inhibition accumulated nuclear p53 and elicited a 25- to 60-fold increase of its transcriptional targets. TP53 regulates MYC, and MDM2/XPO1 inhibition disrupted the c-MYC-regulated transcriptome, resulting in the synergistic induction of apoptosis in acute myeloid leukemia (AML). Unexpectedly, venetoclax-resistant AMLs express high levels of c-MYC and are vulnerable to MDM2/XPO1 inhibition in vivo. However, AML cells persisting after MDM2/XPO1 inhibition exhibit a quiescence- and stress response-associated phenotype. Venetoclax overcomes that resistance, as shown by single-cell mass cytometry. The triple inhibition of MDM2, XPO1, and BCL2 was highly effective against venetoclax-resistant AML in vivo. Our results propose a novel, highly translatable therapeutic approach leveraging p53 reactivation to overcome nongenetic, stress-adapted venetoclax resistance.

Preclinical Evaluation of Bisantrene As Single Agent and in Combination with Decitabine for Acute Myeloid Leukemia

Anthracyclines, particularly doxorubicin and idarubicin, together with cytarabine, have comprised standard of care induction chemotherapy in acute myeloid leukemia (AML) for almost 4 decades. Although 60% of patients achieve remission, the majority relapse within 1-2 years. As such, outcomes in AML remain poor, with overall 5-year survival around 27%. In elderly AML patients the situation is worse, with 80% dying within one year of diagnosis. Toxicity of induction therapy is a major barrier to treatment success, precluding many unfit and elderly patients. Hypomethylating agents (HMA; azacytidine, decitabine) provide a less toxic alternative and have improved treatment options for the unfit. However, only 50% of patients respond to HMA therapy and most eventually acquire resistance, resulting in relapse. Approaches that reduce the toxicity of induction therapy and improve the efficacy of HMA treatment are emerging, e.g. combinations with Venetoclax. Nevertheless, novel combination strategies that improve outcomes in certain mutation subtypes need to be explored. Bisantrene, an anthracene derivative developed over 4 decades ago as a less cardiotoxic chemotherapy alternative to anthracyclines, shows broad activity across multiple solid and haematological malignancies. Several clinical studies in the 1980s showed bisantrene is an effective AML salvage therapy, producing response rates up to 50% without accompanying cardiotoxicity (Rothman J., 2017 Int J Cancer Res Ther;2(2):1-10) In a recent investigator-led Phase II trial of heavily pre-treated relapsed/refractory AML patients, 1/10 achieved a complete remission and 3/10 patients achieved partial remission. Interestingly, all responders were characterised by extramedullary disease (Cannani et al., 2020 Eur J Haematol 2021;106:260-266). We postulate that bisantrene may represent a suitable candidate for combination with HMA in patients unfit for standard induction chemotherapy. To explore this idea, we evaluated the in vitro and in vivo effects of bisantrene both alone and in combination with decitabine. The in vitro activity of bisantrene was initially assessed in a range of human and mouse AML cell lines covering the major molecular and clinical subtypes. All cells were sensitive to bisantrene, showing IC 50 values in the range 16-563 nM. FLT3-ITD + cells in particular were sensitive, while NRAS, KRAS and KIT mutant lines were also responsive. When tested against primary patient AML samples, 15/49 (30.6%) responded strongly to bisantrene treatment ex vivo over the dose range 0.1-1 µM. Bisantrene was shown to induce G1 accumulation and dose-dependent apoptosis in the FLT3-ITD + cell line, MOLM13. When tested in combination with decitabine, pre-treatment with decitabine for 24 h followed by decitabine plus bisantrene for 3 days produced synergistic cytotoxicity in MOLM13, MV4-11 and OCI-AML3 cells. Bisantrene and decitabine together resulted in S phase accumulation in MOLM13 cells and synergistic induction of apoptosis. The in vivo efficacy of bisantrene alone and in combination with decitabine were examined using the MOLM13- luc cell line and a patient derived xenograft (PDX; AML16) (FLT3-ITD +, NPM1, IDH2 and WT1 mutant) in NSG mice. Bisantrene (10 mg/kg i.v. twice/week) significantly reduced leukemic burden and increased median survival compared to vehicle control in MOLM-13- luc engrafted mice, and significantly reduced leukemic burden and increased median survival in a dose and schedule-dependent manner (2.5mg/kg and 5mg/kg, twice or three times weekly) in the PDX model. The combination of bisantrene (5 or 10 mg/kg twice weekly) and decitabine (0.2 mg/kg i.p. 5 days/week) significantly improved survival in MOLM13- luc (Figure 1) and PDX AML16 (Figure 2) engrafted mice, respectively, relative to vehicle control and each single agent. The combination also reduced leukemic infiltration to extramedullary sites (spleen, liver, uterus, brain). These data support that addition of bisantrene to HMA backbone therapy may provide an important new treatment strategy in AML. A prospective Phase Ib/II trial is being planned to evaluate this new therapeutic approach.

A New Class of Synthetic Type-B Polycyclic Polyprenylated Acylphloroglucinols (PPAP-B) Overcomes Venetoclax-Resistance in Acute Myeloid Leukemia (AML)

Venetoclax (VEN) resistance in AML therapy is a field of unmet need. VEN selectively binds in the BH3-binding groove of BCL-2 to release BIM leading to pore-forming BAX/BAK dimerization to finally release cytochrome C, as the “step of no return” in induction of programmed cell death. Compensatory overexpression of other BCL-2 family members such as MCL-1 and BCL-XL is a well described resistance mechanism to restore the ability to sequester BIM and therefore compensate for BCL-2 inhibition. Type-B PPAP are a novel class of neoplastic therapeutics. A new synthetic toolbox for the coupling of a bioactive head group (R 3) enabled the generation of novel, highly active PPAPs, which are low-toxic, water-soluble and photochemically stable - and therefore attractive compounds for clinical development. We now provide evidence that PPAP-B possess antileukemic activity in in vitro and ex vivo AML models via induction of the mitochondrial apoptosis pathways. Further, PPAP-B suppress transcription of MCL-1 and BCL-XL - leading to highly synergistic induction of apoptosis in combination with venetoclax, including a Venetoclax-resistant AML model. Employing structure-activity relationship (SAR) studies, newly generated PPAPs (#22-#144) were tested in acute leukemia HL60, Molm14 and OCI-AML-3 cell lines as well as freshly harvested native leukemia blasts from consented patients. Physiologic mononuclear cells (MC) extracted from cord blood or healthy donors were used as controls. PPAPs were modified step-by-step regarding their chemical structure and assessed in dose dilution series to determine antileukemic efficacy of the compounds. The capability to induce apoptosis and inhibit proliferation was assessed, using standard techniques. To shed light on the underlying mechanism of action, Proteome Profiler Arrays (R&D systems) were performed and confocal microscopy was employed to trace specific fluorescent PPAPs within the cell. Together, the SAR approach led to improvement of IC50 rates of PPAP-B ranging from 20 μM to now < 4 μM. Compound #122 was thereby identified as the most potent PPAP with respect to inhibition of proliferation and induction of apoptosis. Genotoxic studies, as evidenced in a thymidine kinase mouse lymphoma assay (MLA), did not point to potential genotoxicity. Confocal microscopy confirmed accumulation of PPAPs in the cytosol and outer mitochondrial layer using fluorescent PPAPs. In line, induction of mitochondrial apoptosis was seen as evidenced by BAX activation, release of cytochrome C and cleavage of caspases 8 and 3. Further, PPAP-B suppressed transcription of MCL-1 and BCL-XL, putatively via inhibition of the JAK-STAT pathway as discussed earlier. These observations suggest a role of PPAPs in overcoming VEN-resistance in AML. Indeed, combination of PPAP #122 with VEN was highly synergistic with regard to induction of apoptosis, already at doses where the single agents did not display pro-apoptotic activity (e.g. PPAP #122 2.5 μM: 10%, VEN 5nM: 5%, PPAP/VEN combination:74% apoptotis). Most interestingly, PPAP #122 displayed strong pro-apoptotic activity in VEN-resistant leukemia models (MOLM14, HL60, OCi-3 models) with a IC50s < 1μM, and in combination with VEN, re-sensitized cells towards Venetoclax in a combination approach. Together, further development of type-B PPAPs to target Venetoclax resistance in AML is a promising approach to overcome a so far unmet clinical need in acute leukemias.

Synergistic induction of apoptosis in liver cancer cells: exploring the combined potential of doxorubicin and XL-888.

Combination therapy has been frequently preferred in treating various types of cancer in recent years. Targeted cancer therapy has also become one of the remarkable treatment modalities. Therefore, the aim of the study to investigate its cytotoxic and apoptotic effects on liver cancer cell lines by combining the classical chemotherapeutic drug doxorubicin (DOX) and a targeted agent, the new generation HSP90 inhibitor XL-888. The molecular docking method was used to predict the binding conformation of XL-888 on the human Hsp90. The single and combined cytotoxic effects of DOX and XL-888 on liver cancer cell lines HepG2 and HUH-7 were determined by MTT assay. The effect of the combined use of two drugs was evaluated using Chou and Talalay method. The levels of apoptotic genes and heat shock proteins gene and protein expression levels were investigated by quantitative real-time polymerase chain reaction and western blotting, respectively. Molecular docking results showed that XL-888 selectively binds to the ATP binding pocket of HSP90 with an estimated free binding energy of -7.8kcal/mol. DOX and XL-888 and their combination showed dose-dependent cytotoxic effect. The combination of drugs showed a synergistic effect on both cell lines. The results revealed that the combination of DOX and XL-888 potently induced apoptosis in liver cancer cell lines rather than using drugs alone. The combined treatment of DOX and XL-888 demonstrated synergistic cytotoxic and apoptotic effects on liver cancer cell lines, presenting a promising approach for combination therapy in liver cancer.

Oridonin Synergistically Enhances the Pro-Apoptotic Effect of Venetoclax on Acute Myeloid Leukemia Cells by Inhibiting AKT Signaling.

Acute myeloid leukemia (AML) is a recurrence-prone hematologic malignancy. The advent of molecularly targeted therapies provides new opportunities to enhance the effectiveness of AML treatments. Venetoclax, a selective inhibitor of the anti-apoptotic protein Bcl-2, has shown promising results; however, resistance often arises due to elevated expression of the Mcl-1 protein, among other factors. Overcoming this resistance to improve therapeutic outcomes is a pressing issue that requires further investigation. Studies have demonstrated that oridonin, by inhibiting AKT signaling that regulates Mcl-1 expression, can effectively suppress tumor cell growth. This study aims to investigate whether oridonin can synergistically enhance the anti-leukemic effects of venetoclax and explore the underlying mechanisms behind this effect. In vitro experiments were performed to evaluate the effects of the combination of oridonin and venetoclax on AML cell proliferation, apoptosis, cell cycle distribution, and mitochondrial membrane potential. Transcriptome sequencing was used to elucidate the molecular mechanisms underlying the synergistic induction of AML cell apoptosis by the combination therapy. Western blotting and reverse transcription quantitative polymerase chain reaction (RT-qPCR) techniques were used to validate the findings. Additionally, an AML mouse model was established to observe the synergistic anti-AML effects of venetoclax combined with oridonin in vivo. Both venetoclax and oridonin individually exhibited inhibitory effects on AML cell proliferation, resulted in cell cycle arrest, and induced cell apoptosis. Moreover, combination of the two drugs resulted in a synergistic effect. We also observed that oridonin inhibited AKT phosphorylation, upregulated the expression of Bim and Bax proteins, facilitated Mcl-1 degradation, and enhanced the apoptotic effects of venetoclax in AML cells. Finally, in vivo experiments demonstrated that the combination of oridonin and venetoclax effectively inhibited the growth of AML xenograft tumors in mice and prolonged the survival time of tumor-bearing mice. Oridonin and venetoclax synergistically promote AML cell apoptosis by inhibiting AKT signaling.

Abstract 3878: Targeting Hippo-YAP, BRD4 and RAS-MAPK interplay in lung cancer to forestall drug resistance

Abstract Introduction: Identifying parallel survival pathways or compensatory mechanisms that limit the response to targeted therapy is critical to discover effective combinatorial strategies for the treatment of lung cancer and to prevent emergence of drug resistance Results: Using combination drug screening in a panel of cell line models of non-small cell lung cancer (NSCLC), we identified the tumor suppressor STK11 (aka LKB1) as a molecular biomarker of sensitivity to BET and RAS-MAPK pathway inhibitors combination in NSCLC. We found a small molecule inhibitor of BET (bromodomain and extra terminal) family proteins (BRD4, 3, 2), PLX51107, suppressed BRAF and KRAS mutant NSCLC cell proliferation and in combination with RAS (KRAS G12C), RAF or MEK inhibitor led to pronounced loss in cell viability with potent MYC downregulation and synergistic apoptosis induction in vitro and potent tumor regression or stasis in vivo in several KRAS mutant CDX and PDX mouse models of NSCLC. However, BRAF and KRAS mutant NSCLC cells with co-occurring mutations in STK11 were resistant to BET and RAS, RAF or MEK co-inhibition. To unravel the underlying mechanism of resistance to BET and RAS-MAPK inhibitors upon STK11 loss we modeled STK11 deficiency in BRAF and KRAS mutant NSCLC cells and tumors. We found that STK11 loss creates an unprecedented dependence on YAP/TEAD TFs. STK11 silencing in BRAF and KRAS mutant NSCLC cells resulted in nuclear transfer/activation of the HIPPO pathway effector, YAP, upregulation of the YAP target BIRC5 (aka Survivin) and rescued the synthetic lethality due to BET and RAS, RAF or MEK co-inhibition. Phenocopying STK11 loss, constitutively active YAP (YAP5SA), but not transcriptionally inactive YAP (YAPS94A), mitigated the deleterious effects of BET and RAS, RAF or MEK co-suppression in BRAF and KRAS mutant NSCLC cells with potent upregulation of the pro-survival factor BIRC5. Conversely, we found that co-silencing YAP or TEADs with BETs re-sensitized BRAF and KRAS mutant NSCLC cells with co-occurring mutations in STK11 to BET and RAS-MAPK inhibitors combination and caused synthetic lethality. Conclusion: This study uncovered a functional interaction between BET proteins, STK11, HIPPO-YAP and RAS-MAPK signaling pathways in lung cancer. These results will help identify combinatorial strategies for treating aberrant RAS-MAPK pathway driven lung cancer more effectively and forestall drug resistance. Citation Format: Nilanjana Chatterjee, Victor Olivas, Wei Wu, Ben Powell, Trever Bivona. Targeting Hippo-YAP, BRD4 and RAS-MAPK interplay in lung cancer to forestall drug resistance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3878.

Biodegradable nanoplatform upregulates tumor microenvironment acidity for enhanced cancer therapy via synergistic induction of apoptosis, ferroptosis, and anti-angiogenesis

Chemodynamic therapy of cancer is limited by insufficient endogenous H2O2 generation and acidity in the tumor microenvironment (TME). Herein, we developed a biodegradable theranostic platform (pLMOFePt-TGO) involving composite of dendritic organosilica and FePt alloy, loaded with tamoxifen (TAM) and glucose oxidase (GOx), and encapsulated by platelet-derived growth factor-B (PDGFB)-labeled liposomes, that effectively uses the synergy among chemotherapy, enhanced chemodynamic therapy (CDT), and anti-angiogenesis. The increased concentration of glutathione (GSH) present in the cancer cells induces the disintegration of pLMOFePt-TGO, releasing FePt, GOx, and TAM. The synergistic action of GOx and TAM significantly enhanced the acidity and H2O2 level in the TME by aerobiotic glucose consumption and hypoxic glycolysis pathways, respectively. The combined effect of GSH depletion, acidity enhancement, and H2O2 supplementation dramatically promotes the Fenton-catalytic behavior of FePt alloys, which, in combination with tumor starvation caused by GOx and TAM-mediated chemotherapy, significantly increases the anticancer efficacy of this treatment. In addition, T2-shortening caused by FePt alloys released in TME significantly enhances contrast in the MRI signal of tumor, enabling a more accurate diagnosis. Results of in vitro and in vivo experiments suggest that pLMOFePt-TGO can effectively suppress tumor growth and angiogenesis, thus providing an exciting potential strategy for developing satisfactory tumor theranostics.

Inhibition of PRMT5 Increases Sensitivity to BH3 Mimetics in Aggressive B Cell Malignancies

Introduction: Protein Arginine Methyltransferase 5 (PRMT5) is a type II methyltransferase that catalyzes symmetric dimethylation of arginine. PRMT5 is highly expressed in several types of aggressive B cell malignancies, and increased PRMT5 expression has been associated with poor disease prognosis. Several small molecule inhibitors targeting PRMT5 have been developed and showed promising anti-tumor effect in pre-clinical studies including in mantle cell lymphoma (MCL) and diffuse large B cell lymphoma (DLBCL). JNJ-64619178, a potent and specific PRMT5 inhibitor, exhibited broad antiproliferative activity in cancer cell lines from multiple cancer types and cell lines derived xenograft mouse model (Brehmer et al., 2021). In a phase 1 clinical trial of JNJ-64619178 (NCT03573310), patients with advanced solid tumors and non-Hodgkin lymphoma demonstrated manageable toxicity and preliminary evidence of antitumor activity at selected dose levels (Villar et al., 2020). In addition, in patients with lower-risk myelodysplastic syndromes, JNJ-64619178 had manageable hematologic toxicity at the selected expansion dose, but did not have significant clinical activity (Haque et al., 2021). Inhibition of PRMT5 alone could induce cell cycle arrest instead of apoptosis depending on the types of cancer (Zhu et al., 2019), therefore we hypothesized that the combination of PRMT5i and pro-apoptotic BH3 mimetics therapies could be synergistic in aggressive B cell malignancies. Methods: We used dynamic BH3 profiling (DBP) to measure the net pro-apoptotic signaling induced by ex vivo drug treatments (as per Montero et al., 2015). Other standard techniques included shRNA mediated gene knockdown, Western blot, cell viability assays including PI/Annexin V and CellTiter-Glo, and reactive oxygen species (ROS) measurement by flow cytometry with dihydroethidium (DHE) staining. The DLBCL cell lines (HBL1, TMD8, RI-1, OCI-Ly1, Karpas 422, SUDHL4, Toledo), double hit lymphoma (DHL) patient-derived xenograft (PDX) cell lines (DW19), mantle cell lymphoma (MCL) cell lines (Mino, Jeko-1), T cell acute leukemia (T-ALL) cell line (Jurkat) and Burkitt lymphoma (BL) cell line (Raji) were used to investigate the in vitro anti-cancer activity of JNJ-64619178 (Janssen Pharmaceuticals) and BH3 mimetics (venetoclax [BCL2i], S63845 [MCL-1i] and A1331852 [BCL-xLi], Selleckchem). SynergyFinder was used to calculate drug synergism. Results: In all 7 DLBCL cell lines and the DHL-PDX cell line, we found synergistic induction of apoptosis with JNJ-64619178 plus venetoclax (synergy score, 2.82~51.42). The lowest level of synergy was observed in OCI-Ly1 cells, which are intrinsically highly sensitive to venetoclax monotherapy. The combination of JNJ-64619178 with S63845 synergistically induced apoptosis in 5 out of 7 cell lines (synergy score, -1.91~35.64). While the combination of JNJ-64619178 with A1331852 synergistically induced apoptosis in all 7 cell lines (synergy score 1.88~46.73), 4 of them had weak synergy score (1.88~9.4). To confirm the effects we observed with pharmacologic inhibition of PRMT5, we performed shRNA mediated knockdown of PRMT5, which similarly revealed increased sensitivity to venetoclax. In MCL cell lines, combining JNJ-64619178 with venetoclax or A1331852 was more synergistic in inducing cell death than combining with S63845. The T-ALL Jurkat cell line was resistant to JNJ-64619178 and single BH3 mimetic-induced apoptosis. Combining JNJ-64619178 with A1331852 but not venetoclax or S63845 strongly induced apoptosis. The Burkitt cell line Raji was resistant to JNJ-64619178, venetoclax and A1331852, but was sensitive to S63845. The addition of JNJ-64619178 and a single BH3 mimetic in these Raji cells had only a minor synergistic effect. The synergy scores are summarized in Figure 1. Using DBP, JNJ-64619178 was found to increase overall apoptotic priming of DLBCL and MCL cell lines (> 15% increase in cytochrome c loss). By measuring ROS levels, we found increased ROS production (> 2-fold increase in median fluorescent intensity) in response to JNJ-64619178 before the onset of apoptosis, suggesting a possible mechanism by which JNJ-64619178 increases apoptotic priming. Conclusions: The combination of a PRMT5 inhibitor with BH3 mimetics, especially venetoclax, is worthy of further exploration as a potential therapeutic strategy for DLBCL and MCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Venetoclax Resistance in B-Cell Precursor Acute Lymphoblastic Leukemia Is Characterized By Increased Mitochondrial Metabolism and Can be Overcome By Co-Targeting Oxidative Phosphorylation

Deregulated cell death pathways are a hallmark of many cancers and contribute to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Apoptosis is controlled by different pro- and anti-apoptotic molecules either inducing or counteracting cell death induction. Inhibition of anti-apoptotic molecules like B-cell lymphoma 2 (BCL-2) has been developed as therapeutic strategy. Venetoclax (VEN) binds selectively to anti-apoptotic BCL-2 resulting in apoptosis induction and has shown clinical activity in lymphoid malignancies, but insensitivity and resistance have been described. In BCP-ALL, we and others have shown preclinical activity of VEN and VEN is evaluated in first clinical trials. In this study, we have addressed and modeled VEN resistance in BCP-ALL and investigated underlying mechanisms in cell lines and patient-derived xenograft (PDX) samples in order to identify potential strategies to overcome VEN insensitivity. Starting from the BCP-ALL cell line RS4;11, five VEN insensitive lines were generated in parallel by exposure to increasing concentrations of VEN over time (49 passages, 8 months of continuous treatment) and five control lines were exposed to corresponding concentrations of solvent (DMSO). Measuring half maximal effective concentrations (EC50) showed increasing EC50 values from 4 nM to 26.2 µM over time in all VEN treated lines, indicating acquired resistance in our model. VEN insensitive leukemia lines displayed unchanged proliferation and same sensitivities to the prototypic apoptosis inducer staurosporine, the induction therapy drugs vincristine/dexamethasone/asparaginase or the anthracycline daunorubicin as compared to controls, indicating acquisition of VEN specific resistance. In order to further characterize the mechanism of VEN insensitivity, we performed RNA-Seq analysis comparing five RS4;11 VEN insensitive to five control lines. Interestingly, gene set enrichment analysis on significantly regulated genes showed a significant upregulation of gene sets annotated to the citric/tricarboxylic acid cycle and the respiratory electron transport chain, pointing to increased mitochondrial metabolism in VEN resistant cells. We addressed mitochondrial activity and carried out metabolic profiling (Agilent Seahorse XF Cell Mito Stress Test) of VEN resistant and control lines (N=5+5). Most interestingly, in contrast to significant reduction of oxygen consumption rates (OCR) in VEN treated control lines, we found sustained high mitochondrial metabolism indicated by persistent high OCR in VEN resistant lines. To further validate our findings, we investigated a series of BCP-ALL PDX samples (N=30) and titrated VEN EC50 values identifying heterogenous VEN activities. Metabolic profiling (Mito Stress test) was performed on sensitive as compared to highly resistant leukemias (N=3+3). Most interestingly and in line with our findings in cells with acquired resistance, PDX samples with intrinsic VEN insensitivity showed higher oxygen consumption and ATP production rates, thus further highlighting that increased mitochondrial activity is a characteristic feature of VEN resistant ALL. Along this line, significant higher mitochondrial DNA content was found in highly insensitive PDX leukemias when compared to VEN sensitive samples. Moreover, we analyzed mitochondrial morphology by electron microscopy. Although numbers of mitochondria did not differ between VEN resistant and sensitive leukemias, the overall mitochondrial area and structure was found to be significantly larger and elongated, further corroborating our finding of augmented mitochondrial metabolism in VEN resistant ALL. Finally, we addressed whether VEN resistance in ALL can be overcome by directly targeting oxidative phosphorylation (OxPhos). Using oligomycin, an inhibitor of the complex V/ATPase subunit, we found synergistic induction of apoptosis together with VEN in BCP-ALL cell lines and PDX samples, showing that acquired and intrinsic VEN insensitivity can be overcome by co-targeting BCL-2 and the OxPhos pathway. Taken together, we show that VEN resistance in BCP-ALL is characterized by high mitochondrial metabolic activity. Co-targeting of BCL-2 and oxidative phosphorylation results in synergistic anti-leukemia activity setting the basis for further preclinical and potential clinical evaluation.

A Histone Deacetylase Inhibitor Manifests Synergistic Interaction with Artesunate by Suppressing DNA Repair Activity

Artesunate (ART), a plant based semi-synthetic antimalarial drug, is emerging as a new class of effective cancer chemotherapeutics. However, the dosage of ART required to have an anti-cancer effect on cancer cells is greater than that needed to exterminate malarial parasites. The goal of this study was to develop an effective combination therapy to reduce the dose-dependent side effects of ART both in vitro and in vivo. In our study, 4-phenylbutyrate (4-PB), a histone deacetylase inhibitor (HDAC), exhibited significant synergistic induction of apoptosis in MCF-7 cells in combination with ART. The IC50 of ART decreased significantly from 55.56 ± 5.21 µM to 24.71 ± 3.44 µM in MCF-7 cells. ART treatment increased cellular oxidative stress, and the resulting generation of intracellular reactive oxygen species (ROS) caused extensive DNA damage in the cell. The extent of ROS production and cell cycle arrest were further enhanced by 4-PB treatment. In further investigation, we found that 4-PB attenuated mRNA expression of crucial DNA damage response (DDR) elements of the nonhomologous end-joining (NHEJ) pathway, consequently enhancing the DNA damaging effect of ART. Furthermore, the combination therapy resulted in improvement in the life expectancy of the treated mice and a prominent reduction in tumour volume without interfering with the normal biochemical, haematological and histological parameters of the mice. Overall, our study revealed a novel combination therapy in which 4-PB potentiated the cytotoxicity of ART synergistically and provided a promising combination drug for effective cancer therapy.

Beta-naphthoflavone and doxorubicin synergistically enhance apoptosis in human lung cancer cells by inducing doxorubicin accumulation, mitochondrial ROS generation, and JNK pathway signaling

Doxorubicin is one of the most effective chemotherapeutic agents available for treating various cancers, including lung cancer—the leading cause of cancer death in both men and women. However, its clinical application has been impeded by severe adverse effects, notably cardiotoxicity. Development of cellular resistance to doxorubicin is another major obstacle that must be overcome for broader application of the drug. In the present study, we examined the therapeutic potential of beta-naphthoflavone (BNF), a synthetic derivative of a naturally occurring flavonoid, in combination with doxorubicin for the treatment of lung cancer. Among our novel observations were that BNF enhances the efficacy of doxorubicin by inducing doxorubicin accumulation, mitochondrial ROS generation, and JNK pathway signaling in lung cancer cells. These combined effects were also evident in many other cancer cell types. BNF further exhibited synergistic induction of apoptosis in lung cancer cells when combined with several other cancer drugs, including irinotecan, cisplatin, and 5-fluorouracil. Our results suggest that BNF can be developed as a promising adjuvant agent for enhancing the efficacy of doxorubicin.

Functional Testing to Characterize and Stratify PI3K Inhibitor Responses in Chronic Lymphocytic Leukemia.

PI3K inhibitors (PI3Ki) are approved for relapsed chronic lymphocytic leukemia (CLL). Although patients may show an initial response to these therapies, development of treatment intolerance or resistance remain clinical challenges. To overcome these, prediction of individual treatment responses based on actionable biomarkers is needed. Here, we characterized the activity and cellular effects of 10 PI3Ki and investigated whether functional analyses can identify treatment vulnerabilities in PI3Ki-refractory/intolerant CLL and stratify responders to PI3Ki. Peripheral blood mononuclear cell samples (n = 51 in total) from treatment-naïve and PI3Ki-treated patients with CLL were studied. Cells were profiled against 10 PI3Ki and the Bcl-2 antagonist venetoclax. Cell signaling and immune phenotypes were analyzed by flow cytometry. Cell viability was monitored by detection of cleaved caspase-3 and the CellTiter-Glo assay. pan-PI3Kis were most effective at inhibiting PI3K signaling and cell viability, and showed activity in CLL cells from both treatment-naïve and idelalisib-refractory/intolerant patients. CLL cells from idelalisib-refractory/intolerant patients showed overall reduced protein phosphorylation levels. The pan-PI3Ki copanlisib, but not the p110δ inhibitor idelalisib, inhibited PI3K signaling in CD4+ and CD8+ T cells in addition to CD19+ B cells, but did not significantly affect T-cell numbers. Combination treatment with a PI3Ki and venetoclax resulted in synergistic induction of apoptosis. Analysis of drug sensitivities to 73 drug combinations and profiling of 31 proteins stratified responders to idelalisib and umbralisib, respectively. Our findings suggest novel treatment vulnerabilities in idelalisib-refractory/intolerant CLL, and indicate that ex vivo functional profiling may stratify PI3Ki responders.

Abstract 3785: ME-344, a novel isoflavone mitochondrial inhibitor, in combination with venetoclax constitutes a new metabolism-targeted approach to overcome resistance to Bcl-2 inhibition and standard of care treatment in AML

Abstract Acute myeloid Leukemia (AML) is an aggressive hematologic malignancy with poor prognosis. Despite chromosomal and genetic heterogeneity, AML are uniformly characterized by increased reliance on oxidative phosphorylation (OXPHOS). This key metabolic hallmark of leukemia was recently reported as a feature of resistance to Cytarabine (AraC)-based therapy. Also, an aggressive phenotype and poor response to chemotherapy is associated with increased levels of Bcl-2. Despite the introduction of the Bcl-2 inhibitor venetoclax (VEN), the overall survival, particularly in older patients, remains poor. Thus, approaches to improve the sensitivity of leukemic cells to AraC-based or Bcl-2 based therapies are urgently needed.Here, we investigated the preclinical activity of ME-344, a novel isoflavone OXPHOS inhibitor, on AML cell lines and relapsed/refractory (R/R) patient samples in vitro and examined the efficacy of ME-344 in combination with VEN in Ara-C sensitive and resistant AML cell lines and patient-derived xenografts (PDX) both in vitro and in vivo.ME-344 (0-300 nM, 24 hr) significantly reduced viability of AML cell lines with EC50 of 75-100 nM and R/R AML patient samples with EC50 of 200-300 nM respectively. The cytotoxic response in AML was enhanced when ME-344 was combined with VEN, producing strong synergistic viability reduction and induction of apoptosis, as evidenced by Annexin V assay and an increased level of cleaved caspase 3 and PARP (immunoblotting). The dual inhibition of OXPHOS/Bcl-2 reduced Mcl-1 levels and showed efficacy in Mcl-1 overexpressingand Ara-C resistant AML models.Functional metabolic characterization of AML by transcriptomics and mass spectrometry demonstrated that ME-344 effectively inhibited biosynthetic pathways of nucleotides uncovering the purine biosynthesis pathway as crucial for therapeutic efficacy. ME-344 induced a dose-dependent decrease in the oxygen consumption rate (by Seahorse assay), in both AraC-sensitive and -resistant AML cell lines, and in R/R AML patient samples, which was further significantly potentiated by combination with VEN.Finally, in an aggressive AML xenograft model, ME-344 (200 mpk, i.v.) combined with subtherapeutic doses of VEN (25 mpk) reduced circulating leukemia burden and extended survival. Ongoing in vivo studies in AML PDX models will address the impact of ME-344 in the context of acquired AraC- and Bcl-2- resistance. In summary, our findings indicate ME-344 alone or in combination with Bcl-2 inhibition constitutes an important therapeutic modality that targets a unique metabolic vulnerability of AML. Citation Format: Katie H. Hurrish, Yongwei Su, Shraddha Patel, Sandra E. Wiley, Zhanjun Hou, Jenna Carter, Hasini Kalpage, Maik Hüttemann, Connie Weng, Holly Edwards, Lisa Polin, Jing Li, Jay Yang, Larry H. Matherly, Sergej Naumovich Konoplev, Jeffrey W. Taub, Marina Konopleva, Yubin Ge, Natalia Baran. ME-344, a novel isoflavone mitochondrial inhibitor, in combination with venetoclax constitutes a new metabolism-targeted approach to overcome resistance to Bcl-2 inhibition and standard of care treatment in AML [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 3785.

Targeted liposomes for combined delivery of artesunate and temozolomide to resistant glioblastoma

The effective treatment of glioblastoma (GBM) is a great challenge because of the blood-brain barrier (BBB) and the growing resistance to single-agent therapeutics. Targeted combined co-delivery of drugs could circumvent these challenges; however, the absence of more effective combination drug delivery strategies presents a potent barrier. Here, a unique combination ApoE-functionalized liposomal nanoplatform based on artesunate-phosphatidylcholine (ARTPC) encapsulated with temozolomide (ApoE-ARTPC@TMZ) was presented that can successfully co-deliver dual therapeutic agents to TMZ-resistant U251-TR GBM in vivo. Examination in vitro showed ART-mediated inhibition of DNA repair through the Wnt/β-catenin signaling cascade, which also improved GBM sensitivity to TMZ, resulting in enhanced synergistic DNA damage and induction of apoptosis. In assessing BBB permeation, the targeted liposomes were able to effectively traverse the BBB through low-density lipoprotein family receptors (LDLRs)-mediated transcytosis and achieved deep intracranial tumor penetration. More importantly, the targeted combination liposomes resulted in a significant decrease of U251-TR glioma burden in vivo that, in concert, substantially improved the survival of mice. Additionally, by lowering the effective dosage of TMZ, the combination liposomes reduced systemic TMZ-induced toxicity, highlighting the preclinical potential of this novel integrative strategy to deliver combination therapies to brain tumors.

The BCL-2 inhibitor ABT-199/venetoclax synergizes with proteasome inhibition via transactivation of the MCL-1 antagonist NOXA

Enhanced expression of anti-apoptotic B-cell lymphoma 2 (BCL-2) protein is frequent in cancer. Targeting of BCL-2 with the specific inhibitor ABT-199 (Venetoclax) has significant clinical activity in malignant diseases such as chronic lymphocytic leukemia and multiple myeloma. The small molecule drug ABT-199 mimics the pro-apoptotic BCL-2 homology domain 3 of BH3-only proteins and blocks the hydrophobic BC-groove in BCL-2. We have previously shown that ABT-199 synergizes with the proteasome inhibitor (PI) bortezomib in soft tissue sarcoma derived cells and cell lines to induce apoptosis. Synergistic apoptosis induction relies on the pore-forming effector BAX and expression of the pro-apoptotic BH3-only protein NOXA. Bortezomib augments expression of NOXA by blocking its proteasomal degradation. Interestingly, shown here for the first time, expression of NOXA is strongly enhanced by ABT-199 induced integrated stress response (ISR). ISR transcription factors ATF3 & ATF4 mediate transactivation of the BH3-only protein NOXA which specifically inhibits the anti-apoptotic MCL-1. Thus, NOXA potentiates the efficacy of the BCL-2 inhibitor ABT-199 by simultaneous inhibition of MCL-1. Hence, ABT-199 has a double impact by directly blocking anti-apoptotic BCL-2 and inhibiting MCL-1 via transactivated NOXA. By preventing degradation of NOXA PIs synergize with ABT-199. Synergism of ABT-199 and PIs therefore occurs on several, previously unexpected levels. This finding should prompt clinical evaluation of combinatorial regimens in further malignancies.

Metformin exerts synergistic anti-proliferative effects with liraglutide in human umbilical vein endothelial cells (HUVECs).

Objective(s):Metformin (Met) and liraglutide (Lira) have been approved to treat type 2 diabetes mellitus and have cardioprotective effects. Materials and Methods:Human umbilical vein endothelial cells (HUVECs) were incubated with Met, Lira, or their combination in this study. Results:Results showed that the synergistic inhibitory effect of the two drugs on HUVECs proliferation was significant (75%) after 48 hr drug exposure. In addition, either Lira or Met alone had a marked tendency to inhibit the migration of HUVECs (42% and 39%). Almost a complete inhibition (97%) was demonstrated in combinational use after 48 hr treatment. After combining these two drugs, the apoptosis rate raised to 68%, which was a significant approval of synergistic apoptosis induction of Met and Lira. The combinational group indicated a substantial increase in VEGF, PDGF, and MMP-9 at 24 hr compared with the control. Conclusion:This study showed that combination therapy with Lira and Met could effectively reduce cell proliferation, induce apoptosis, and inhibit cell migration in the HUVECs. This study provides evidence to support using Met in combination with Lira as a treatment option for patients with type-2 diabetes and cancer.