Synergistic Antiproliferative Activity Research Articles

Peposertib, a DNA-PK Inhibitor, Enhances the Anti-Tumor Efficacy of Topoisomerase II Inhibitors in Triple-Negative Breast Cancer Models.

Triple-negative breast cancer (TNBC) remains the most lethal subtype of breast cancer, characterized by poor response rates to current chemotherapies and a lack of additional effective treatment options. While approximately 30% of patients respond well to anthracycline- and taxane-based standard-of-care chemotherapy regimens, the majority of patients experience limited improvements in clinical outcomes, highlighting the critical need for strategies to enhance the effectiveness of anthracycline/taxane-based chemotherapy in TNBC. In this study, we report on the potential of a DNA-PK inhibitor, peposertib, to improve the effectiveness of topoisomerase II (TOPO II) inhibitors, particularly anthracyclines, in TNBC. Our in vitro studies demonstrate the synergistic antiproliferative activity of peposertib in combination with doxorubicin, epirubicin and etoposide in multiple TNBC cell lines. Downstream analysis revealed the induction of ATM-dependent compensatory signaling and p53 pathway activation under combination treatment. These in vitro findings were substantiated by pronounced anti-tumor effects observed in mice bearing subcutaneously implanted tumors. We established a well-tolerated preclinical treatment regimen combining peposertib with pegylated liposomal doxorubicin (PLD) and demonstrated strong anti-tumor efficacy in cell-line-derived and patient-derived TNBC xenograft models in vivo. Taken together, our findings provide evidence that co-treatment with peposertib has the potential to enhance the efficacy of anthracycline/TOPO II-based chemotherapies, and it provides a promising strategy to improve treatment outcomes for TNBC patients.

Discovery of Novel Heterotricyclic Compounds as DNA-Dependent Protein Kinase (DNA-PK) Inhibitors with Enhanced Chemosensitivity, Oral Bioavailability, and the Ability to Potentiate Cancer Immunotherapy.

In this work, a novel series of heterotricyclic DNA-PK inhibitors were rationally designed, synthesized, and assessed for their biological activity. In the DNA-PK biochemical assay, most compounds displayed potent enzymatic activity, with IC50 values between 0.11 and 71.5 nM. Among them, SK10 exhibited the most potent DNA-PK-inhibitory activity (IC50 = 0.11 nM). Studies of the mechanism of action indicated that SK10 could lower γH2A.X expression levels and demonstrate optimal synergistic antiproliferative activity against Jurkat cells (IC50 = 25 nM) when combined with doxorubicin. Importantly, in CT26 and B16-F10 tumor-bearing mouse models, the combination therapies of SK10 with chemotherapeutic drug doxorubicin, a PD-L1 antibody, and SWS1 (a potent PD-L1 small-molecule inhibitor) demonstrated superior synergistic anticancer and potential immunomodulatory effects. Furthermore, SK10 possessed favorable in vivo pharmacokinetic properties [e.g., oral bioavailability (F) = 31.8%]. Taken together, SK10 represents a novel heterotricyclic DNA-PK inhibitor with antitumor immune effects and favorable pharmacokinetics.

Abstract 4745: A novel combination therapy targeting RAF, MEK and FAK to overcome skin cutaneous melanoma treatment resistance

Abstract Metastatic melanoma is the most aggressive malignancy of the skin and BRAFV600E mutation is the most common genetic alteration in skin cutaneous melanoma (SKCM). Despite the advent of immune checkpoint inhibition (ICI) immunotherapy, only 40% of patients show long-term responses. As such, combined therapy with BRAF and MEK inhibitors (BRAFi + MEKi) remains the standard of care for BRAFV600E+ SKCM. In the present study, transcriptome analysis of BRAFV600E+ SKCM tumors derived from patients revealed that activation of extracellular matrix signaling, including focal adhesion signaling, is highly enriched in patients who experienced disease progression on BRAFi + MEKi therapy. Consistent with these results, we found increased activation of focal adhesion kinase (FAK) in human BRAFV600E+ SKCM A375 cells treated with BRAFi, MEKi or the RAF/MEK clamp avutometinib. Mechanistically, we discovered that avutometinib-mediated inhibition of the RAF/MEK/ERK pathway decreased RhoE/Rnd3 expression, thereby unleashing RhoA/FAK/AKT signaling. Thus, we hypothesized that FAK activation represents a resistance mechanism to BRAFi + MEKi and that FAK inhibition (FAKi) might overcome resistance to BRAFi/MEKi. Indeed, avutometinib demonstrated synergistic antiproliferative and proapoptotic activity when combined with FAKi in human BRAFV600E+ SKCM A375 cells. Importantly, we found that the combination of FAKi + avutometinib overcame resistance to BRAFi + MEKi in SKCM xenografts and patient-derived cells from SKCM-resistant lesions. Finally, we showed that while BRAFV600E+ SKCM YUMM 1.7 syngeneic tumors failed to respond to ICI therapy, avutometinib ± FAKi inhibited tumor growth. We observed that tumors treated with avutometinib as single agent eventually developed resistance and escaped growth inhibition, but those treated with combined avutometinib and FAKi displayed durable treatment responses, often with complete tumor regression. These findings provide a rationale for the clinical development of avutometinib with defactinib (FAKi) in patients with BRAFV600E+ cutaneous melanoma following progression on BRAFi + MEKi and/or ICI therapy. Citation Format: Simone Lubrano, Farhoud Faraji, Rodolfo Daniel Cervantes-Villagrana, Sydney Ramirez, Nadia Arang, Adam Officer, Damiano C. Rigiracciolo, Paola Y. Anguiano Quiroz, Antonieta Bacchiocchi, Ruth Halaban, Silvia Coma, Sheri Holmen, Claudia Martini, Jonathan Pachter, Andrew Aplin, J. Silvio Gutkind. A novel combination therapy targeting RAF, MEK and FAK to overcome skin cutaneous melanoma treatment resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4745.

Overcoming doxorubicin resistance in triple-negative breast cancer using the class I-targeting HDAC inhibitor bocodepsin/OKI-179 to promote apoptosis

BackgroundTriple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with a poor prognosis. Doxorubicin is part of standard curative therapy for TNBC, but chemotherapy resistance remains an important clinical challenge. Bocodepsin (OKI-179) is a small molecule class I histone deacetylase (HDAC) inhibitor that promotes apoptosis in TNBC preclinical models. The purpose of this study was to investigate the combination of bocodepsin and doxorubicin in preclinical TNBC models and evaluate the impact on terminal cell fate, including apoptosis and senescence.MethodsTNBC cell lines were treated with doxorubicin and CellTiter-Glo was used to assess proliferation and determine doxorubicin sensitivity. Select cell lines were treated with OKI-005 (in vitro version of bocodepsin) and doxorubicin and assessed for proliferation, apoptosis as measured by Annexin V/PI, and cell cycle by flow cytometry. Immunoblotting was used to assess changes in mediators of apoptosis, cell cycle arrest, and senescence. Senescence was measured by the senescence-associated β-galactosidase assay. An MDA-MB-231 xenograft in vivo model was treated with bocodepsin, doxorubicin, or the combination and assessed for inhibition of tumor growth. shRNA knockdown of p53 was performed in the CAL-51 cell line and proliferation, apoptosis and senescence were assessed in response to combination treatment.ResultsOKI-005 and doxorubicin resulted in synergistic antiproliferative activity in TNBC cells lines regardless of p53 mutation status. The combination led to increased apoptosis and decreased senescence. In vivo, the combination resulted in increased tumor growth inhibition compared to either single agent. shRNA knock-down of p53 led to increased doxorubicin-induced senescence that was decreased with the addition of OKI-005 in vitro.ConclusionThe addition of bocodepsin to doxorubicin resulted in synergistic antiproliferative activity in vitro, improved tumor growth inhibition in vivo, and promotion of apoptosis which makes this a promising combination to overcome doxorubicin resistance in TNBC. Bocodepsin is currently in clinical development and has a favorable toxicity profile compared to other HDAC inhibitors supporting the feasibility of evaluating this combination in patients with TNBC.

FPFT-2216, a Novel Anti-lymphoma Compound, Induces Simultaneous Degradation of IKZF1/3 and CK1α to Activate p53 and Inhibit NFκB Signaling.

We found potential vulnerability to CK1α degradation in certain lymphoma cells refractory to IKZF1/3 degraders. Targeting CK1α with FPFT-2216 could inhibit the growth of these cells by activating p53 signaling. Our study demonstrates the potential therapeutic application of CK1α degraders, such as FPFT-2216, for treating lymphoma.

Antiproliferative Activity of Piceamycin by Regulating Alpha-Actinin-4 in Gemcitabine-Resistant Pancreatic Cancer Cells.

Although gemcitabine-based regimens are widely used as an effective treatment for pancreatic cancer, acquired resistance to gemcitabine has become an increasingly common problem. Therefore, a novel therapeutic strategy to treat gemcitabine-resistant pancreatic cancer is urgently required. Piceamycin has been reported to exhibit antiproliferative activity against various cancer cells; however, its underlying molecular mechanism for anticancer activity in pancreatic cancer cells remains unexplored. Therefore, the present study evaluated the antiproliferation activity of piceamycin in a gemcitabine-resistant pancreatic cancer cell line and patient-derived pancreatic cancer organoids. Piceamycin effectively inhibited the proliferation and suppressed the expression of alpha-actinin-4, a gene that plays a pivotal role in tumorigenesis and metastasis of various cancers, in gemcitabine-resistant cells. Long-term exposure to piceamycin induced cell cycle arrest at the G0/G1 phase and caused apoptosis. Piceamycin also inhibited the invasion and migration of gemcitabine-resistant cells by modulating focal adhesion and epithelial-mesenchymal transition biomarkers. Moreover, the combination of piceamycin and gemcitabine exhibited a synergistic antiproliferative activity in gemcitabine-resistant cells. Piceamycin also effectively inhibited patient-derived pancreatic cancer organoid growth and induced apoptosis in the organoids. Taken together, these findings demonstrate that piceamycin may be an effective agent for overcoming gemcitabine resistance in pancreatic cancer.

PSPC1 Inhibition Synergizes with Poly(ADP-ribose) Polymerase Inhibitors in a Preclinical Model of BRCA-Mutated Breast/Ovarian Cancer.

Poly (ADP-ribose) polymerase (PARP) inhibitors are effective against BRCA1/2-mutated cancers through synthetic lethality. Unfortunately, most cases ultimately develop acquired resistance. Therefore, enhancing PARP inhibitor sensitivity and preventing resistance in those cells are an unmet clinical need. Here, we investigated the ability of paraspeckle component 1 (PSPC1), as an additional synthetic lethal partner with BRCA1/2, to enhance olaparib sensitivity in preclinical models of BRCA1/2-mutated breast and ovarian cancers. In vitro, the combined olaparib and PSPC1 small interfering RNA (siRNA) exhibited synergistic anti-proliferative activity in BRCA1/2-mutated breast and ovarian cancer cells. The combination therapy also demonstrated synergistic tumor inhibition in a xenograft mouse model. Mechanistically, olaparib monotherapy increased the expressions of p-ATM and DNA-PKcs, suggesting the activation of a DNA repair pathway, whereas combining PSPC1 siRNA with olaparib decreased the expressions of p-ATM and DNA-PKcs again. As such, the combination increased the formation of γH2AX foci, indicating stronger DNA double-strand breaks. Subsequently, these DNA-damaged cells escaped G2/M checkpoint activation, as indicated by the suppression of p-cdc25C (Ser216) and p-cdc2 (Tyr15) after combination treatment. Finally, these cells entered mitosis, which induced increased apoptosis. Thus, this proves that PSPC1 inhibition enhances olaparib sensitivity by targeting DNA damage response in our preclinical model. The combination of olaparib and PSPC1 inhibition merits further clinical investigation to enhance PARP inhibitor efficacy.

The Novel Profiling Relative Inhibition Simultaneously in Mixtures (PRISM) Platform Identifies Synergistic Activity of Lanraplenib and Ruxolitinib in Hematological Malignancies

Background: Investigation of drug combinations across different contexts can provide useful insights on the anti-cancer mechanism and can ultimately lead to new treatments. However, conventional drug combination screening methods are limited by throughput. Efforts to systematically identify the most effective active combinations and the optimal molecular contexts in a high throughput screening (HTS) format could greatly accelerate the development of combination treatments. Spleen Tyrosine Kinase, SYK, is a non-receptor tyrosine kinase known to regulate intracellular signaling, including FLT3, AKT/mTOR and STAT5 pathways, via its immunoreceptor tyrosine-based activation motif (ITAM). Deregulated SYK signaling has been reported to play a central role in pathogenesis of allergic and autoimmune diseases as well as hematological malignancies. Lanraplenib (LANRA) is a next-generation SYK inhibitor currently being evaluated in combination with gilteritinib, a FLT3 inhibitor, in patients with relapsed or refractory (R/R) FLT3-mutated acute myeloid leukemia (AML) (NCT05028751). Given its critical role in intracellular signaling and interaction with receptor tyrosine kinases(RTKs), we hypothesized that LANRA could synergize with ruxolitinib, a JAK inhibitor. To address this hypothesis, we performed a high throughput drug combination screen using the Broad Institute's Profiling Relative Inhibition Simultaneously in Mixtures (PRISM) platform, which enables rapid screening of thousands of compounds in a 930-cell line panel across 45 different lineages. Methods: In this study, we performed a combination screen using the PRISM platform with ruxolitinib as the test compound, with a 10 μM top dose (7 dose concentrations with 3-fold dilutions), in combination with LANRA at two anchor doses, 2 μM and 10 μM. PRISM is a pooled, multiplexed cell viability assay that provides 7-point dose response curves, IC 50, AUC values, and relative abundance of unique cell line barcodes. To understand the drug synergy landscape across different lineages, we developed a bioinformatics pipeline which uses PRISM viability data to calculate synergy scores across all the cell lines and drug combinations. Secondary validation studies of the combinations used CTG viability measurements. Phospho-SYK expression was evaluated in archival formalin-fixed paraffin-embedded (FFPE) bone marrow biopsies from patients with myeloid proliferative neoplasm (MPN) by immunohistochemistry (IHC). RNA-seq was performed to evaluate differential changes in gene expression in response to LANRA. Gene set enrichment analysis (GSEA) was performed to evaluate perturbation in leukemogenic signaling pathways. Results: In the PRISM cell line panel, ruxolitinib in combination with LANRA demonstrated synergistic activity in hematological malignancy cell lines. Among AML cell lines in the panel, OCIAML5, OCIM1, HL60, HEL, EOL1, MONOMAC6, NB4, U937, PL21, and TF1 showed the highest synergy scores. The most sensitive cell lines to the combination showed up-regulation of JAK-STAT and inflammatory signaling pathways in a gene set enrichment analysis (GSEA) prior to treatment. Consistent with this, phosphorylated SYK was associated with inflammatory megakaryocytes and fibrosis in primary samples from patients with MPN. LANRA down-regulated JAK-STAT signaling in a reporter cell line and repressed gene expression associated with dysregulated inflammatory pathways in AML cells. Additionally, the combination of LANRA and ruxolitinib showed synergistic antiproliferative activity across a broad range of concentrations in FLT3-ITD primary AML cells and a panel of hematological malignancy cell lines, confirming the PRISM results. Conclusion: These studies demonstrate the utility of PRISM as a platform to rapidly identify rational combination agents. Importantly, LANRA is effective in combination with ruxolitinib in AML and other hematological malignancy preclinical models. This finding is consistent with the observation that SYK can regulate STAT signaling and cooperate with other RTKs like FLT3. Given the central role of SYK in regulation of oncogenic and inflammatory signaling, SYK inhibition with LANRA in combination with ruxolitinib may be a promising strategy for patients with myeloid malignancies.

C-type natriuretic peptide (CNP) inhibits 7,12-Dimethylbenz[a]anthracene (DMBA)/Croton oil-induced skin tumor growth by modulating inflammation in Swiss albino mice.

C-type natriuretic peptide (CNP) exhibits anti-inflammatory activity besides its natriuretic and diuretic functions. The present study aimed to determine the anticancer and synergistic therapeutic activity of CNP against a 7,12-Dimethylbenz[a]anthracene (DMBA)/Croton oil-induced skin tumor mouse model. CNP (2.5 µg/kg body weight) was injected either alone and/or in combination with Cisplatin (CDDP) (2 mg/kg body weight) for 4 weeks. The dorsal skin tumor incidences/growth and mortality rate were recorded during the experimental period of 16 weeks. The serum C-reactive protein (CRP), and lactate dehydrogenase (LDH) levels, infiltrating mast cells, and AgNORs proliferating cells count were analyzed in control and experimental mice. Further, the expression profile of marker genes of proliferation, inflammation, and progression molecules were analyzed using Reverse transcriptase-polymerase chain reaction(RT-PCR)/quantitative PCR(qPCR), western blot, and immunohistochemistry. The DMBA/Croton oil-induced mice exhibited 100% tumor incidence. Whereas, CNP alone, CDDP alone, and CNP+CDDP combination-treated mice exhibited 58%, 46%, and 24% tumor incidence, respectively. Also, a marked reduction in the levels of serum CRP and LDH, the number of infiltrating mast cells count and AgNORs proliferating cells count were noticed in the mice skin sections. Further, a significant reduction in both mRNA and protein expression levels of proliferation, inflammation, and progression markers were noticed in CNP (p < 0.01), CDDP (p < 0.01), and CNP+CDDP combination (p < 0.001) treated mice, respectively. The results of the present study suggest that CNP has anticancer activity. Further, the CNP+CDDP treatment has more promising anticancer activity as compared with CNP or CDDP alone treatment, probably due to the synergistic antiproliferative and anti-inflammatory activities of CNP and CDDP.

High-Throughput Drug Screen for Potential Combinations With Venetoclax Guides the Treatment of Transformed Follicular Lymphoma.

Transformed follicular lymphoma (t-FL) is an aggressive malignancy that is refractory and rapidly progressing with poor prognosis. There is currently no effective treatment. High-throughput screening (HTS) platforms are used to profile the sensitivity or toxicity of hundreds of drug molecules, and this approach is applied to identify potential effective treatments for t-FL. We randomly selected a compound panel from the School of Pharmaceutical Sciences Xiamen University, tested the effects of the panel on the activity of t-FL cell lines using HTS and the CCK-8 assay, and identified compounds showing synergistic anti-proliferative activity with the Bcl-2 inhibitor venetoclax (ABT-199). Bioinformatics tools were used to analyze the potential synergistic mechanisms. The single-concentration compound library demonstrated varying degrees of activity across the t-FL cell lines evaluated, of which the Karpas422 cells were the most sensitive, but it was the cell line with the least synergy with ABT-199. We computationally identified 30 drugs with synergistic effects in all cell lines. Molecularly, we found that the targets of these 30 drugs didn't directly regulate Bcl-2 and identified 13 medications with high evidence value above .9 of coordination with ABT-199, further confirming TP53 may play the largest role in the synergistic effect. Collectively, these findings identified the combined regimens of ABT-199 and further suggested that the mechanism is far from directly targeting Bcl-2, but rather through the regulation and synergistic action of p53 and Bcl-2. This study intended to reveal the best synergistic scheme of ABT-199 through HTS to more quickly inform the treatment of t-FL.

A phase 3, two-stage, randomized study of mezigdomide, carfilzomib, and dexamethasone (MeziKd) versus carfilzomib and dexamethasone (Kd) in relapsed/refractory multiple myeloma (RRMM): SUCCESSOR-2.

TPS8070 Background: Mezigdomide (MEZI), a novel oral cereblon E3 ligase modulator (CELMoD), induces maximal degradation of Ikaros/Aiolos leading to increased MM cell apoptosis and immune-stimulatory effects. MeziKd has shown potent synergistic antiproliferative activity in MM cell lines resistant to lenalidomide (LEN), and in a phase 1/2 study showed promising preliminary efficacy and safety in RRMM. The SUCCESSOR-2 phase 3 trial (NCT05552976) will compare the efficacy and safety of MeziKd vs Kd in patients (pts) with RRMM. Methods: This multicenter, open-label study comprises 2 stages. In Stage 1, ≥ 128 pts will be randomized 3:3:3:2 to 1 of 3 MEZI doses (1.0, 0.6, or 0.3 mg) + Kd, or to the Kd arm to select the optimal MEZI dose in combination with Kd for Stage 2. In Stage 2, ≈ 397 additional pts will be randomized 3:2 to MeziKd at the selected MEZI dose or to Kd, for efficacy and safety analyses (Stage 1 pts in the selected MeziKd dose cohort and Kd arm will also be included in these analyses). Pts will be stratified by age (≤ 70 vs &gt; 70 y), number of prior lines of treatment (Tx; ≤ 2 vs &gt; 2), and ISS stage at screening (I vs II vs III). MEZI dose selection will be based on Stage 1 efficacy, safety, pharmacokinetic and pharmacodynamic data, and exposure–response analyses. Primary efficacy endpoint is progression-free survival (PFS). Assuming a decrease in PFS risk by 33.3% (HR = 0.667) with MeziKd, under exponential distribution assumption of PFS (1-sided α = 0.025) and adjusted for 3 interim analyses, ≥ 273 PFS events will have ≈ 89% power to detect improvement in Tx effect. The planned interim analyses are for: MEZI dose selection at end of Stage 1, and to examine PFS futility and superiority when ≈ 82 (30%) and ≈ 205 (75%) events, respectively, have been accumulated. Secondary endpoints include determination of the recommended MEZI dose plus Kd (Stage 1 only), and assessment of overall survival, overall response rate, time to response, duration of response, time to progression, safety, and quality of life. MeziKd arm Tx consists of 28-day (D) cycles (C) with MEZI on D1–21; 20 mg/m2 intravenous (IV) carfilzomib (CFZ) on D1 of C1, then 56 mg/m2 on D8 and 15 of C1, on D1, 8, and 15 of C2–12, and on D1 and 15 of ≥ C13; and 40 mg oral/IV DEX (20 mg optional in certain pt groups) on D1, 8, 15, and 22. Tx in the Kd arm consists of 28-D cycles with 20 mg/m2 IV CFZ on D1 and 2 of C1, then 56 mg/m2 on D8, 9, 15, and 16 of C1, and on D1, 2, 8, 9, 15, and 16 of ≥ C2; and 20 mg oral/IV DEX (10 mg optional in certain pt groups) on D1, 2, 8, 9, 15, 16, 22, and 23. Tx will continue until progressive disease (PD) or unacceptable toxicity. Key eligibility criteria include age ≥ 18 y, ≥ 1 prior line of anti-MM Tx including LEN and an anti-CD38 monoclonal antibody, minimal response or better to ≥ 1 prior Tx, documented PD during or after last regimen, and no prior CFZ Tx. Enrollment began in October 2022 and is ongoing. Clinical trial information: NCT05552976 .

Abstract 6215: Peposertib, a DNA-PK inhibitor, enhances the antitumor efficacy of anthracyclines in triple-negative breast cancer models in vitro and in vivo

Abstract Background: Triple-negative breast cancer (TNBC) is the most lethal breast cancer subtype, exhibiting poor response rates toward current chemotherapy regimens and lacking additional effective treatment options. Approximately 30% of patients with TNBC respond well to anthracycline/taxane-based standard-of-care chemotherapy regimens. However, remaining patients demonstrate limited improvements in clinical outcomes, highlighting the critical need for effective strategies to improve anthracycline/taxane-based chemotherapy (1, 2). DNA-dependent protein kinase (DNA-PK), a member of the phosphoinositide 3-kinase-related kinase protein family, promotes nonhomologous end joining (NHEJ) as part of the DNA damage response. Peposertib is a potent, selective, and orally bioavailable DNA-PK inhibitor that strongly potentiates the antitumor effects of ionizing radiation and DNA double-strand break-inducing agents, including anthracyclines, in preclinical models. Here we report the synergistic antitumor effects of peposertib combined with topoisomerase II (TOPO II) inhibitors, particularly anthracyclines, in TNBC models in vitro and in vivo. Methodology: Combinations of peposertib with TOPO II inhibitors were evaluated in TNBC cell lines by viability assays, immunoblotting, and flow cytometry. Gene expression analysis was performed using the nCounter PanCancer pathways panel and results were confirmed by quantitative polymerase chain reaction (qPCR). In vivo efficacy was assessed in cell-line derived and patient-derived xenograft models. Results: When administered in combination with doxorubicin, epirubicin, and etoposide, peposertib exhibited synergistic antiproliferative activity in TNBC cell lines in vitro. The downstream analysis of pharmacodynamic biomarkers revealed induction of NHEJ mediated repair upon TOPO II inhibitor treatment and provided mechanistic insights into the synergistic antitumor effects of peposertib combined with TOPO II inhibitors. Furthermore, this combination treatment induced ataxia telangiectasia mutated (ATM)-dependent compensatory signaling and inflammatory responses, potentially creating a proinflammatory tumor microenvironment. To evaluate whether peposertib enhanced the antitumor effects of TOPO II inhibitors in vivo, we established a well-tolerated preclinical treatment schedule for the combined use of peposertib and pegylated liposomal doxorubicin, which was capable of achieving tumor regression in patient- and cell line-derived xenograft models of TNBC. Conclusion: Our findings suggest that cotreatment with the DNA-PK inhibitor peposertib can enhance the efficacy of anthracycline/TOPO II-based chemotherapies. This work was supported by the healthcare business of Merck KGaA, Darmstadt, Germany (CrossRef Funder ID: 10.13039/100009945). 1) Davison C et al. NPJ Breast Cancer. 2021;7(1):38. 2) Bianchini G et al. Nat Rev Clin Oncol. 2016;13(11):674-690. Citation Format: Steffie Revia, Christian Sirrenberg, Antonia Schach, Astrid Zimmermann, Frank T. Zenke, Joachim Albers. Peposertib, a DNA-PK inhibitor, enhances the antitumor efficacy of anthracyclines in triple-negative breast cancer models in vitro and in vivo. [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 6215.

Abstract 3461: Preclinical and clinical evaluation of the RAF/MEK clamp avutometinib (VS-6766) in combination with the mTOR inhibitor everolimus for the treatment of KRAS mutated non-small cell lung cancer

Abstract Background: KRAS mutations (mt) are found in ~30% of non-small cell lung cancers (NSCLC). Despite the approval and development of KRAS G12C and KRAS G12D inhibitors respectively, mechanisms of resistance to MAPK pathway inhibitors are emerging and combination strategies are needed for patients with KRAS mt NSCLC . Avutometinib (VS-6766) is a unique RAF/MEK clamp that inhibits MEK kinase activity and blocks RAF-mediated phosphorylation of MEK. We studied the preclinical and clinical activity of the combination of avutometinib with the mTOR inhibitor everolimus in KRAS mt NSCLC. Methods: A panel of KRAS mt NSCLC cell lines were treated for 1 hour with clinically relevant concentrations of avutometinib and everolimus and changes in phosphoproteins were measured using an antibody array. We then tested for synergy of avutometinib and everolimus in 3D proliferation assays and in the H441 NSCLC xenograft model. A clinical trial is ongoing (NCT02407509) testing the combination of 3.2 mg avutometinib with 5 mg of everolimus administered twice weekly 3 weeks on/1 week off in 28-day cycles (previously defined as the recommended phase 2 dose) in a cohort of patients with KRAS mt NSCLC. Results: Avutometinib inhibited the MAPK pathway (p-MEK, p-ERK, p-90RSK) with an increase in p-PRAS40, suggesting activation of the PI3K pathway as an adaptive resistance mechanism. Everolimus inhibited the PI3K pathway (p-p70S6K and p-RPS6). Among a panel of KRAS mt NSCLC cell lines, avutometinib + everolimus showed synergistic anti-proliferative activity across KRAS G12C, G12V and G12D variants (mean synergy score of ~18). In the H441 KRAS G12V NSCLC xenograft model, there was a significant reduction in tumor volume and increase in survival with the combination of avutometinib and everolimus (87% TGI; 66 days vs 36.5 days in control group) compared to control. In the clinical trial expansion, 16 patients with KRAS mt NSCLC have been treated so far with avutometinib and everolimus (5 G12V, 3 Q61H, 2 G12C, 2 G12A, 2 G12D, 1 G13A, 1 G13D; median prior lines = 2). The current objective response rate (ORR) among the 14 patients who have at least one post assessment scan is 3/14 (21%; 1 G12V, 1 G12A, 1 G13A) with 11/14 showing a reduction in tumor size as best response. The current progression free survival (PFS) is 5.3 months (95% CI 2.8-7.4 months) with 4 patients still on study. Updated data on the planned cohort size of 20 patients will be presented. Conclusion: The combination of avutometinib and everolimus overcomes the activation of the PI3K/AKT/mTOR pathway which is an adaptive resistance mechanism to MAPK pathway inhibition. We have shown that avutometinib and everolimus induce synergistic anti-tumor effects preclinically, and preliminary data suggest clinically meaningful ORR and PFS in patients with KRAS mt NSCLC including non-G12C variants. Citation Format: Simon Rodney, Adam R. Stewart, Victoria Sanchez Perez, Cienne Morton, Lisa A. Pickard, Taleen Shakouri, Toby Prout, Mona Parmar, Alison J. Turner, Silvia Coma, Jonathan Pachter, Laura Finneran, Emma Hall, James Spicer, Anna Minchom, Udai Banerji. Preclinical and clinical evaluation of the RAF/MEK clamp avutometinib (VS-6766) in combination with the mTOR inhibitor everolimus for the treatment of KRAS mutated non-small cell lung cancer [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 3461.

Abstract P4-07-04: STX-478, a mutant-selective PI3Kα H1047X inhibitor clinical candidate with a best-in-class profile: Pharmacology and therapeutic activity as monotherapy and in combination in breast cancer xenograft models

Abstract PI3Kα is highly mutated in cancer resulting in hyperactivation of lipid kinase activity and downstream AKT signaling. H1047 is the most common site of oncogenic mutation and occurs in ~14% of all breast cancers. Initial therapeutic benefit of targeting PI3Kα was established with alpelisib, an alpha-selective PI3K inhibitor that is equipotent against wild-type and mutant forms. However, wild-type PI3Kα inhibition results in frequent dose-limiting toxicities including hyperglycemia, restricting the full potential of this drug. Selective targeting of H1047X-mutant PI3Kα is expected to both improve anti-tumor activity and reduce toxicity. STX-478 is an allosteric, CNS-penetrant, selective PI3Kα H1047X inhibitor, having excellent drug-like properties and exceptional kinome selectivity. STX-478 demonstrated minimal inhibition of CYP enzymes in vitro, supporting the potential for combinations with a wide range of therapeutics in breast cancer and a variety of other tumor types. STX-478 selectivity extended to the inhibition of other activating kinase domain mutations in biochemical assays. In a diverse panel of PI3Kα H1047X mutant cell lines, STX-478 selectively reduced the cellular levels of pAKT (S473) with a strong correlation between pAKT inhibition and cell viability (R = 0.8). In a high-throughput viability screen of 467 cancer cell lines, the presence of PIK3CA H1047X and other kinase domain mutations were the single strongest predictor of STX-478 sensitivity with potency superior to alpelisib. STX-478 also selectively inhibited the proliferation of cell lines with PI3Kα helical domain mutations, potentially due to the selective dependency of these cells on mutant PI3Kα. When combined with fulvestrant, lapatinib, or abemaciclib, STX-478 demonstrated synergistic anti-proliferative activity in cell lines with relevant ER/HER2 status. Unlike alpelisib, STX-478 did not impair glucose metabolism or cause insulin resistance at efficacious doses. In the T47D (PI3Kα H1047R) breast cancer model, STX-478 (100 mg/kg) monotherapy caused tumor regression whereas alpelisib caused only stasis. STX-478 combination with fulvestrant was well-tolerated, with more consistent and deeper tumor regression. Similar results were observed in a PI3Kα H1047R mutant ER+/HER2- PDX model, where fulvestrant monotherapy showed minimal activity, while combination with STX-478 yielded tumor regressions. In an ER+/HER2+ PDX model (PI3Kα H1047R/R108H), palbociclib and STX-478 (100 mg/kg) monotherapy resulted in similar efficacy while the combination was well tolerated and yielded tumor regression. Together these data indicate robust STX-478 monotherapy activity that was well tolerated and improved when dosed in combination with fulvestrant or CDK4/6 inhibitors. Finally, we investigated the effect of STX-478 treatment in an ER+ PDX model carrying a helical domain mutation. STX-478 treatment resulted in tumor growth inhibition at doses that did not result in metabolic dysfunction, suggesting that STX-478 may also be efficacious in treating PIK3CA mutant tumors with helical domain mutations. In summary, STX-478 efficacy was superior to alpelisib at a dose level that exceeds the clinically relevant exposure in mice without causing metabolic dysfunction. STX- 478 has a predicted low human dose, CNS exposure, low risk of DDI, and a predicted long half-life with minimal variation in peak-to-trough plasma concentrations which further supports a favorable therapeutic index. STX-478 has the potential to provide a best-in-class profile to improve outcomes in patients harboring tumors with prevalent PI3Kα H1047X mutations as well as other kinase and helical domain mutant tumors. The significant CNS exposure of STX-478 is expected to enable this treatment for patients with brain tumors and brain metastases not afforded by existing options. STX-478 is currently in IND enabling studies and is expected to enter human clinical trials in 2023. Citation Format: Leonard Buckbinder, David J. St. Jean, Brendon Ladd, Trang Tieu, Philip Jonsson, Jacob Alltucker, Samantha Manimala, Weixue Wang, Angel Guzman-Perez, Darrin D. Stuart, Gregory Dowdell. STX-478, a mutant-selective PI3Kα H1047X inhibitor clinical candidate with a best-in-class profile: Pharmacology and therapeutic activity as monotherapy and in combination in breast cancer xenograft models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-07-04.

Inhibition of autophagy and MEK promotes ferroptosis in Lkb1-deficient Kras-driven lung tumors

LKB1 and KRAS are the third most frequent co-mutations detected in non-small cell lung cancer (NSCLC) and cause aggressive tumor growth. Unfortunately, treatment with RAS-RAF-MEK-ERK pathway inhibitors has minimal therapeutic efficacy in LKB1-mutant KRAS-driven NSCLC. Autophagy, an intracellular nutrient scavenging pathway, compensates for Lkb1 loss to support Kras-driven lung tumor growth. Here we preclinically evaluate the possibility of autophagy inhibition together with MEK inhibition as a treatment for Kras-driven lung tumors. We found that the combination of the autophagy inhibitor hydroxychloroquine (HCQ) and the MEK inhibitor Trametinib displays synergistic anti-proliferative activity in KrasG12D/+;Lkb1-/- (KL) lung cancer cells, but not in KrasG12D/+;p53-/- (KP) lung cancer cells. In vivo studies using tumor allografts, genetically engineered mouse models (GEMMs) and patient-derived xenografts (PDXs) showed anti-tumor activity of the combination of HCQ and Trametinib on KL but not KP tumors. We further found that the combination treatment significantly reduced mitochondrial membrane potential, basal respiration, and ATP production, while also increasing lipid peroxidation, indicative of ferroptosis, in KL tumor-derived cell lines (TDCLs) and KL tumors compared to treatment with single agents. Moreover, the reduced tumor growth by the combination treatment was rescued by ferroptosis inhibitor. Taken together, we demonstrate that autophagy upregulation in KL tumors causes resistance to Trametinib by inhibiting ferroptosis. Therefore, a combination of autophagy and MEK inhibition could be a novel therapeutic strategy to specifically treat NSCLC bearing co-mutations of LKB1 and KRAS.

Synergistic anticancer effect of combination treatment of vitamin D and pitavastatin on the HCC1937 breast cancer cells

Vitamin D (Vit D) has anticancer properties including activating cell senescence inhibiting cancer cell proliferation, inducing apoptotic cell death, and decreasing cancer cell migration. On the other hand, statins showed favorable anticancer activities including anti-survival, anti-proliferation, and anti-migration effects. The current study aimed to investigate the synergistic anticancer effect of Vit D and statins against HCC1937 triple-negative breast cancer cells. The antiproliferative effect was tested by MTT assay after 48 hours of the treatments. Trypan blue test and clonogenic assay were used to test the anti-survival activities of the treatments. The ability of the treatments to inhibit the migration ability was tested by scratch assay. Levels of the cell cycle and apoptotic markers were determined by western blotting. Results of the study revealed that all the tested compounds including Vit D, atorvastatin (Ator), simvastatin (Simv), and pitavastatin (Pita) inhibited HCC1937 breast cancer cell growth with different IC50 values ranging from 4.49-12.95 µM. Combined application of Pita and Vit D showed potent synergistic antiproliferative activities against HCC1937 breast cancer cells. The combined therapy of (1µM Vit D and 2 µM Pita) inhibited HCC1937 cell proliferation by cell cycle arrest and apoptosis as evidenced by increasing p21, p53, and cleaved PARP. Finally, the combined treatment decreased the p-STAT3 level in HCC1937 breast cancer cells. The results of the study can be concluded that the combined treatment of Pita and Vit D has a synergistic anticancer effect against HCC1937 breast cancer cells.

SYK Inhibitors, Entospletinib and Lanraplenib, Show Potent Anti-Leukemic Activity in Combination with Targeted Agents

Introduction: Spleen tyrosine kinase (SYK) acts as a key integrator of cellular signaling from surface contact and receptor tyrosine kinase receptors containing an immunoreceptor tyrosine-based activation motif (ITAM). In acute myeloid leukemia (AML), SYK serves as a relay to an oncogenic transcriptional regulatory network (TRN) linked to NPM1, HOXA9, and MEIS1 helping to suppress myeloid progenitor maturation and promote proliferation. The selective, oral SYK inhibitor, entospletinib (ENTO), has demonstrated clinical activity in HOXA9/MEIS1-driven AML with acceptable tolerability when combined with intensive induction chemotherapy. A global phase 3 trial (AGILITY, NCT05020665) is currently underway, evaluating the combination of ENTO with intensive induction/consolidation chemotherapy in patients with treatment-naive NPM1-mutated (NPM1m) AML with measurable residual disease as the primary endpoint. Lanraplenib (LANRA) is a next-generation SYK inhibitor with similar potency, enhanced selectivity, and more favorable pharmacologic properties that is currently being evaluated in combination with gilteritinib in patients with relapsed or refractory (R/R) FLT3-mutated AML (NCT05028751). Results: ENTO exhibits anti-proliferative activity in NPM1m AML with associated HOXA9/MEIS1 dysregulation in ex vivo drug sensitivity studies. We hypothesized that SYK inhibition could synergize with other targeted therapies to enhance anti-leukemic activity at different nodes in this pathway. To address this hypothesis, the FLT3 internal tandem duplication/MLL-rearranged cell lines, MOLM13 and MV411, were treated with increasing doses of either LANRA or ENTO in combination with a small molecule MLL-Menin inhibitor (SNDX5613). Synergistic anti-proliferative effects were observed across a broad range of concentrations. No combinatorial or single-agent SNDX5613 activity was seen in the KASUMI-1 AML cell line, which lacks an MLL rearrangement. Phenotypic studies using flow cytometry were performed to assess the mechanistic response for SYK inhibition with LANRA in combination with SNDX5613. LANRA was found to increase the percentage of CD11b-positive cells and mean fluorescence intensity, while SNDX5613 led to a marked increase in CD14. In contrast to either single agent, the combination triggered early apoptosis and cell death, suggesting a more complete blockade of the HOXA9/MEIS1 transcriptional program through synergistic inhibition by orthogonal mechanisms. Furthermore, treatment of leukemic cells with either ENTO or LANRA inhibited SYK auto-phosphorylation in a dose-dependent manner. We also explored the potential for synergistic activity with gilteritinib (FLT3 inhibitor) and venetoclax (BCL2 inhibitor) using patient-derived AML isolates ex vivo based on the potential for SYK signaling cross talk with FLT3 itself and survival pathways. We found strong synergistic anti-proliferative activity for the combination of LANRA with either gilteritinib or venetoclax. Patient-derived xenograft (PDX) studies also demonstrated deeper reductions in leukemic burden in the peripheral blood, liver, and bone marrow after 28 days of treatment. Follow-up PDX studies assessing overall survival and pharmacodynamic activity are ongoing using an optimized dosing regimen and schedule. Summary/Conclusion: SYK is a promising therapeutic target for genetically defined subsets of AML. Two highly selective SYK inhibitors are currently undergoing clinical testing in different but complementary clinical settings. Given its central role as a modulator of leukemogenic signaling, SYK inhibition has the potential to synergize with other targeted therapies in AML. In preclinical studies, LANRA-mediated SYK inhibition shows compelling anti-proliferative and pro-apoptotic activity in combination with gilteritinib, venetoclax, and SNDX5613 in genetically defined subsets of AML.

Lisaftoclax in Combination with Alrizomadlin Overcomes Venetoclax Resistance in Acute Myeloid Leukemia and Acute Lymphoblastic Leukemia: Preclinical Studies.

Despite approval of B-cell lymphoma (BCL)-2 inhibitor venetoclax for certain hematologic malignancies, its broader clinical benefit is curtailed by resistance. Our study aimed to determine if treatment with novel anticancer agents targeting BCL-2 and mouse double minute 2 (MDM2) could overcome venetoclax resistance in preclinical models. Venetoclax-sensitive and venetoclax-resistant acute myeloid leukemia (AML) and acute lymphoblastic leukemia cells and xenograft models were used to evaluate antitumor effects and underlying mechanisms associated with combined BCL-2 inhibitor lisaftoclax (APG-2575) and MDM2 inhibitor alrizomadlin (APG-115). The combination exhibited synergistic antiproliferative and apoptogenic activities in TP53 wild-type AML cell lines in vitro. This synergy was further exemplified by deep antitumor responses and prolonged survival in AML cell line-derived and patient-derived xenograft models. Interestingly, the combination treatment resensitized (to apoptosis) venetoclax-resistant cellular and mouse models established via chronic drug exposure or genetically engineered with clinically relevant BCL-2 gene mutations. Synergistic effects in reducing cellular viability and proliferation were also demonstrated in primary samples of patients with venetoclax-resistant AML treated with lisaftoclax and alrizomadlin ex vivo. Mechanistically, alrizomadlin likely primes cancer cells to BCL-2 inhibition-induced cellular apoptosis by downregulating expression of antiapoptotic proteins myeloid cell leukemia-1 and BCL-extra-large and upregulating pro-death BCL-2-associated X protein. Lisaftoclax in combination with alrizomadlin overcomes venetoclax resistance mediated by various mechanisms, including BCL-2 mutations. In addition, we posit further, putative molecular mechanisms. Our data rationalize clinical development of this treatment combination in patients with diseases that are insensitive or resistant to venetoclax.

Rational drug combination design in patient-derived avatars reveals effective inhibition of hepatocellular carcinoma with proteasome and CDK inhibitors

BackgroundHepatocellular carcinoma (HCC) remains difficult to treat due to limited effective treatment options. While the proteasome inhibitor bortezomib has shown promising preclinical activity in HCC, clinical trials of bortezomib showed no advantage over the standard-of-care treatment sorafenib, highlighting the need for more clinically relevant therapeutic strategies. Here, we propose that rational drug combination design and validation in patient-derived HCC avatar models such as patient-derived xenografts (PDXs) and organoids can improve proteasome inhibitor-based therapeutic efficacy and clinical potential.MethodsHCC PDXs and the corresponding PDX-derived organoids (PDXOs) were generated from primary patient samples for drug screening and efficacy studies. To identify effective proteasome inhibitor-based drug combinations, we applied a hybrid experimental-computational approach, Quadratic Phenotypic Optimization Platform (QPOP) on a pool of nine drugs comprising proteasome inhibitors, kinase inhibitors and chemotherapy agents. QPOP utilizes small experimental drug response datasets to accurately identify globally optimal drug combinations.ResultsPreliminary drug screening highlighted the increased susceptibility of HCC PDXOs towards proteasome inhibitors. Through QPOP, the combination of second-generation proteasome inhibitor ixazomib (Ixa) and CDK inhibitor dinaciclib (Dina) was identified to be effective against HCC. In vitro and in vivo studies demonstrated the synergistic pro-apoptotic and anti-proliferative activity of Ixa + Dina against HCC PDXs and PDXOs. Furthermore, Ixa + Dina outperformed sorafenib in mitigating tumor formation in mice. Mechanistically, increased activation of JNK signaling mediates the combined anti-tumor effects of Ixa + Dina in HCC tumor cells.ConclusionsRational drug combination design in patient-derived avatars highlights the therapeutic potential of proteasome and CDK inhibitors and represents a feasible approach towards developing more clinically relevant treatment strategies for HCC.

Ipatasertib, an oral AKT inhibitor, in combination with carboplatin exhibits anti-proliferative effects in serous endometrial cancer: Endometrial Cancer Molecularly Targeted Therapy Consortium (238)

<b>Objectives:</b> Ipatasertib (IPAT) is an investigational, orally-administered, ATP-competitive, highly selective inhibitor of protein kinase B (AKT) that has demonstrated antitumor activity in metastatic prostate cancer and longer progression-free survival in triple-negative breast cancer. Hyperactivation of the PI3K/AKT signaling pathway is frequently seen in endometrial cancer (EC), including serous histology tumors. Thus, we evaluated the antiproliferative efficacy of IPAT in combination with standard-of-care carboplatin in human serous EC cell lines. <b>Methods:</b> The human serous EC cell lines, ARK-1 and SPEC-2, were exposed to varying concentrations of IPAT (Genetech) and carboplatin. Cell proliferation was assessed by MTT assay. To assess the synergy of IPAT and carboplatin, the combination index (CI) was calculated by the method of Chou and Talalay. Apoptosis was assessed by cleaved caspase-3 assay. Cellular stress was measured by DCFDA assay. Western immunoblotting determined the effects of IPAT and carboplatin on apoptotic (MCL-1 and BCL-2) and cellular stress (Bip and PERK) pathways in the ARK-1 and SPEC-2 cell lines. <b>Results:</b> IPAT and carboplatin significantly inhibited cell proliferation in a dose-dependent manner after 72 hours of treatment. The median IC₅₀ for IPAT was 8µM in the ARK-1 and 5µM in SPEC-2. The median IC₅₀ for carboplatin was 50µM in the ARK-1 and 65µM in SPEC-2. Simultaneous exposure of cells to various doses of carboplatin in combination with IPAT resulted in a significant synergistic antiproliferative effect (CI<1). IPAT and carboplatin induced the activity of cleaved-caspase-3 by 2.3-fold in ARK-1 and 3.8-fold in SPEC-2 (p<0.05) while simultaneously reducing expression of the apoptotic proteins MCL-1 and BCL-2. DCFDA assay analysis demonstrated that IPAT and carboplatin induced ROS products by 1.39-fold and 1.17-fold in the ARK-1 and SPEC-2 cell lines (p<0.05), respectively, and increased Bip and PERK protein expression. The effects of IPAT combined with carboplatin on apoptosis and cellular stress were greater when compared to IPAT or carboplatin alone in both cell lines (p<0.05). <b>Conclusions:</b> The combination of IPAT and carboplatin showed synergistic antiproliferative activity in human serous EC cell lines. Thus, IPAT plus carboplatin may be a promising combination in the treatment of serous EC. Studies in transgenic and patient-derived xenograft EC mouse models are underway to assess the in vivo efficacy of IPAT +/- carboplatin.