Synergistic Antitumor Activity Research Articles

Using the Iron Chelator Deferasirox to Overcome Drug Resistance in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is a difficult-to-treat B-cell malignancy characterized by the t(11,14) translocation, resulting in cyclin D1 (CD1) overexpression. In addition to CD1 overexpression, pathways such as the B-cell receptor, PI3K/AKT/ GSK3β, NFκB and Wnt have been reported to be deregulated in MCL. Despite advancements in MCL treatment, most patients still relapse. Although the introduction of ibrutinib to relapsed/refractory MCL significantly improved the outcome of MCL patients, ibrutinib resistance has become a clinical obstacle. MCL treatments are thus pursued by studying novel agents with a broad spectrum of targets or by rationally combining existing therapies aiming for synergistic antitumor activities. Deferasirox (DFX) is a clinically approved iron chelator with only few side effects. DFX has been reported to exert anti-tumoral and synergistic effects in several types of cancers by affecting a multitude of targets. We have previously shown that DFX exerts a vigorous anti-tumoral effect via growth inhibition and induction of apoptosis in MCL cells through ROS elevation, triggering of oxidative stress, induction of DNA damage, modulation of PI3K/AKT/GSK3β signaling and most importantly by elimination of CD1 expression. The capacity of DFX to affect a multitude of targets establishes a solid basis for a possible synergistic interaction with other drugs, such that may overcome drug resistance in MCL. In this study we focused on assessing the efficiency of DFX combined with the established therapeutic-agents etoposide, cytarabine and ibrutinib in MCL cell-lines. We found that DFX synergizes with etoposide, cytarabine and ibrutinib, prompting remarkable anti-tumoral effects in MCL cells with combination index (CI) values < 1. Interestingly, the DFX-drug combinations achieved synergism regardless of the innate sensitivity of the cell-lines to the treatment: ibrutinib-resistant cells restored their sensitivity to the drug when it was combined with DFX. In addition, we found that the sensitivity of MCL cells towards the drugs correlated with the drugs ability to induce CD1 degradation. In agreement, DFX co-treatment enhanced CD1 degradation, especially in resistant cells.We show here that DFX is a putative promising drug-sensitizing agent for the treatment of MCL. DFX-co-treatment not only enhances the efficacy of the tested drugs, but also restores the anti-proliferative activity of the drugs in resistant MCL cells. To the best of our knowledge, this study is the first to provide evidence on the potential of DFX to overcome drug resistance in MCL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Safety and Efficacy of CD37-Targeting Naratuximab Emtansine PLUS Rituximab in Diffuse Large B-Cell Lymphoma and Other NON-Hodgkin'S B-Cell Lymphomas - a Phase 2 Study

Safety and Efficacy of CD37-Targeting Naratuximab Emtansine PLUS Rituximab in Diffuse Large B-Cell Lymphoma and Other NON-Hodgkin'S B-Cell Lymphomas - a Phase 2 Study

Synergistic antitumor activity of pan-PI3K inhibition and immune checkpoint blockade in bladder cancer

BackgroundImmune checkpoint blockade (ICB) induces durable response in approximately 20% of patients with advanced bladder urothelial cancer (aUC). Over 50% of aUCs harbor genomic alterations along the phosphoinositide 3-kinase (PI3K)...

Antiangiogenic antibody BD0801 combined with immune checkpoint inhibitors achieves synergistic antitumor activity and affects the tumor microenvironment

BackgroundSignaling through VEGF/VEGFR induces cancer angiogenesis and affects immune cells. An increasing number of studies have recently focused on combining anti-VEGF/VEGFR agents and immune checkpoint inhibitors (ICIs) to treat cancer in preclinical and clinical settings. BD0801 is a humanized rabbit anti-VEGF monoclonal antibody in the clinical development stage.MethodsIn this study, the anti-cancer activities of BD0801 and its potential synergistic anti-tumor effects when combined with different immunotherapies were assessed by using in vitro assays and in vivo tumor models. Ex vivo studies were conducted to reveal the possible mechanisms of actions (MOA) underlying the tumor microenvironment modification.ResultsBD0801 showed more potent antitumor activity than bevacizumab, reflected by stronger blockade of VEGF/VEGFR binding and enhanced inhibitory effects on human umbilical vein endothelial cells (HUVECs). BD0801 exhibited dose-dependent tumor growth inhibitory activities in xenograft and murine syngeneic tumor models. Notably, combining BD0801 with either anti-PD-1 or anti-PD-L1 antibodies showed synergistic antitumor efficacy in both lung and colorectal cancer mouse models. Furthermore, the mechanistic studies suggested that the MOA of the antitumor synergy involves improved tumor vasculature normalization and enhanced T-cell mediated immunity, including increased tumor infiltration of CD8+ and CD4+ T cells and reduced double-positive CD8+PD-1+ T cells.ConclusionsThese data provide a solid rationale for combining antiangiogenic agents with immunotherapy for cancer treatment and support further clinical development of BD0801 in combination with ICIs.

Overcoming Gemcitabine Resistance in Pancreatic Cancer Using the BCL-XL-Specific Degrader DT2216.

Pancreatic cancer is the third most common cause of cancer-related deaths in the United States. Although gemcitabine is the standard of care for most patients with pancreatic cancer, its efficacy is limited by the development of resistance. This resistance may be attributable to the evasion of apoptosis caused by the overexpression of BCL-2 family antiapoptotic proteins. In this study, we investigated the role of BCL-XL in gemcitabine resistance to identify a combination therapy to more effectively treat pancreatic cancer. We used CRISPR-Cas9 screening to identify the key genes involved in gemcitabine resistance in pancreatic cancer. Pancreatic cancer cell dependencies on different BCL-2 family proteins and the efficacy of the combination of gemcitabine and DT2216 (a BCL-XL proteolysis targeting chimera or PROTAC) were determined by MTS, Annexin-V/PI, colony formation, and 3D tumor spheroid assays. The therapeutic efficacy of the combination was investigated in several patient-derived xenograft (PDX) mouse models of pancreatic cancer. We identified BCL-XL as a key mediator of gemcitabine resistance. The combination of gemcitabine and DT2216 synergistically induced cell death in multiple pancreatic cancer cell lines in vitro In vivo, the combination significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice compared with the individual agents in pancreatic cancer PDX models. Their synergistic antitumor activity is attributable to DT2216-induced degradation of BCL-XL and concomitant suppression of MCL-1 by gemcitabine. Our results suggest that DT2216-mediated BCL-XL degradation augments the antitumor activity of gemcitabine and their combination could be more effective for pancreatic cancer treatment.

Phase II Study of Single-Agent and Combination Everolimus and Panobinostat in Relapsed or Refractory Diffuse Large B-Cell Lymphoma

Novel therapeutics are needed for patients with relapsed or refractory diffuse large B-cell lymphoma (R/R DLBCL). Everolimus is an mTOR pathway inhibitor with synergistic anti-tumor activity when combined with histone deacetylase inhibitors, such as panobinostat, in preclinical lymphoma models. In this Phase II study, we evaluated overall response rate to single and combination everolimus and panobinostat in R/R DLBCL. Fifteen patients were enrolled to single-agent and 18 to combination. One patient responded to everolimus, while none responded to panobinostat. Though 25% of patients responded to combination therapy, responses were not durable with significant toxicity. We demonstrated minimal single-agent activity and prohibitive toxicity with combination therapy.

Antitumor immune effects of preoperative sitravatinib and nivolumab in oral cavity cancer: SNOW window-of-opportunity study

BackgroundSitravatinib, a tyrosine kinase inhibitor that targets TYRO3, AXL, MERTK and the VEGF receptor family, is predicted to increase the M1 to M2-polarized tumor-associated macrophages ratio in the tumor microenvironment...

ATHENA (GOG-3020/ENGOT-ov45): a randomized, phase III trial to evaluate rucaparib as monotherapy (ATHENA–MONO) and rucaparib in combination with nivolumab (ATHENA–COMBO) as maintenance treatment following frontline platinum-based chemotherapy in ovarian cancer

BackgroundThe optimal treatment strategy for women with newly diagnosed ovarian cancer has yet to be determined. Poly(ADP-ribose) polymerase (PARP) inhibitors have demonstrated substantial improvement in progression-free survival as monotherapy maintenance...

Combination of Compound Kushen Injection and cisplatin shows synergistic antitumor activity in p53-R273H/P309S mutant colorectal cancer cells through inducing apoptosis

Ethnopharmacological relevanceColorectal cancer (CRC) is one type of worldwide popular and refractory tumors. Compound Kushen Injection (CKI) is a frequently applied traditional Chinese medicine formula as an adjuvant drug for the chemotherapy of CRC. P53 is the most commonly mutated gene in CRC, accounting for the development, malignant and prognosis progression of CRC. However, effect of CKI on the therapeutic efficacy of p53-mutant CRC remains understood. Besides, the combined efficacy of different chemotherapeutics drugs in combination with CKI for CRC treatment is rarely concerned. Aim of studyTo investigate the combined efficacy of the CKI-derived combination strategies in the p53-mutant CRC. Materials and methodsTwo CRC cell lines HCT116 and SW480 cells, which respectively harbor wild-type p53 and p53-R273H/P309S mutant, were applied. Cisplatin (Cis) and 5-fluorouracil (5FU) were combined chemotherapeutics drugs of CKI-derived combination strategies in this article. In vitro antitumor activity was detected by sulforhodamine B (SRB) assay and colony formation assay. Combenefit soft was applied to evaluate the synergetic/antagonistic effect of drug combination. Lentivirus-mediated overexpression method was used to generate a set of p53-mutant and wild-type CRC cell lines harboring identical genomes. Transcriptomics and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were applied to predicate the underlying mechanism of synergetic interaction between drug combination. Western blot was performed to verify predicated pathways contributing to the synergy of drug combination. ResultsCKI preferentially combined with Cis but not 5FU, to produce a synergistical antitumor efficiency for p53-R273H/P309S mutant, rather than wild-type p53 harboring CRC cells. The combination of CKI and Cis strongly reprogrammed the transcriptional profiles of SW480 cells. Cytokine-cytokine receptor interaction pathway was a key pathway involved in cooperativity between CKI and Cis in SW480 cells. Mechanistically, compared to that Cis individually triggered necroptosis, the co-treatment of CKI and Cis reinforced the cell death of SW480 cells in a possible synergistic manner by inducing extrinsic apoptosis pathway. ConclusionThis article provides a novel perspective into the precision clinical application of CKI-derived combination therapy programs of CRC based on genetic variation and the classes of chemotherapeutics drugs.

816TiP Randomized phase III trial on niraparib - TSR-042 (dostarlimab) vs physician’s choice CHEmotherapy in recurrent ovarian, fallopian tube or primary peritoneal cancer patients not candidate for platinum retreatment: NItCHE trial (MITO 33)

Platinum resistant ovarian cancer patients have a poor prognosis and few treatment options are available. Preclinical and clinical data demonstrated that the combination of poly-ADP ribose polymerase inhibitors with immune checkpoint inhibitors could have a synergistic antitumor activity in this setting of patients.

434P CodeBreaK 101 subprotocol H: Phase Ib study evaluating combination of sotorasib (Soto), a KRASG12C inhibitor, and panitumumab (PMab), an EGFR inhibitor, in advanced KRAS p.G12C-mutated colorectal cancer (CRC)

Soto, a specific, irreversible KRASG12C inhibitor, has monotherapy clinical activity in KRAS p.G12C-mutated solid tumors, with an objective response rate of 7.1% for heavily pretreated CRC in the CodeBreaK 100 phase I trial. KRASG12C blockade can lead to accumulation of activated EGFR. Combining Soto with an EGFR inhibitor may produce synergistic antitumor activity. The safety and efficacy of Soto with PMab, a monoclonal antibody specific to EGFR, in KRAS p.G12C-mutated CRC are being evaluated in this ongoing phase Ib study (NCT04185883). This study includes a dose exploration phase to identify a safe/tolerable daily oral dose of Soto with PMab (6mg/kg IV Q2W) in patients (pts) with previously treated mCRC, and a dose expansion phase. Dose exploration is reported. As of 4/23/21, 8 pts (5 female, median age: 60.5 yrs [range: 31-79]) were enrolled in dose exploration with 960 mg QD Soto and 6mg/kg IV Q2W PMab. Median number of lines of therapy for metastatic disease was 3.5 (range 1-10); 5 pts had prior Soto. Median treatment (tx) duration was 4.4 months (range: 1.4, 8.8). No dose limiting toxicities (DLTs) were observed during the DLT evaluation period (first 28 days). Tx-related adverse events (TRAEs) of any grade related to Soto or PMab were reported for 4 and 8 pts, respectively. No grade 4 or fatal TRAEs occurred. Two pts had PMab TRAEs leading to dose modification of PMab (1–dermatitis acneiform, 1–dry skin, rash, hypokalemia, hypomagnesemia) and 1 pt had a Soto TRAE leading to dose modification of Soto (diarrhea). There was 1 confirmed partial response, 5 stable disease (SD), 1 progressive disease (PD), and 1 not evaluated but with clinical PD. Of pts with prior Soto, 4 had decrease in sum of target lesions; 4 had SD and 1 with PD developed new lesions despite a decrease in size of target lesions. Sotorasib exposures were similar to those observed in monotherapy study. Combination of Soto (960 mg QD) and PMab (6mg/kg IV Q2W) was safe and tolerable with promising efficacy in heavily pretreated pts with KRAS p.G12C-mutated CRC. AEs are consistent with known AEs for Soto and PMab. Updated safety and efficacy data will be presented.

MCL-101: Using the Iron Chelator Deferasirox to Overcome Drug Resistance in Mantle Cell Lymphoma

<h3>Context</h3> Mantle cell lymphoma (MCL) is a difficult-to-treat B-cell malignancy characterized by the t(11;14) translocation, resulting in cyclin D1 (CD1) overexpression. Despite advancements in MCL treatment, most patients still relapse. For instance, although the introduction of ibrutinib for relapsed/refractory MCL significantly improved the outcomes of MCL patients, ibrutinib resistance has become a clinical obstacle. MCL treatments are thus pursued by studying novel agents with a broad spectrum of targets or by rationally combining existing therapies aiming for synergistic anti-tumor activities. Deferasirox (DFX) is a clinically approved iron chelator with little to no side effects. We have previously shown that DFX exerts a vigorous anti-tumoral effect via growth inhibition and induction of apoptosis in MCL cells through ROS elevation, triggering of oxidative stress, induction of DNA damage, modulating PI3K/AKT/GSK3β signaling, and most importantly eliminating CD1 expression. The capacity of DFX to affect a multitude of targets establishes a solid basis for a possible synergistic interaction with other drugs, such that may overcome drug resistance in MCL. <h3>Objectives</h3> This study focuses on assessing the efficiency of DFX combined with the established therapeutic-agents etoposide, cytarabine, and ibrutinib in MCL cell-lines. <h3>Results</h3> We found that DFX synergizes with etoposide, cytarabine, and ibrutinib, prompting remarkable anti-tumoral effects in MCL cells with combination index (CI) values < 1. Interestingly, the DFX-drug combinations achieved synergism regardless of the innate sensitivity of the cell-lines to the treatment: ibrutinib-resistant cells restored their sensitivity to the drug when it was combined with DFX. In addition, we found that the sensitivity of MCL cells toward the drugs correlated with the drugs' ability to induce CD1 degradation. In agreement, DFX co-treatment enhanced CD1 degradation, especially in resistant cells. <h3>Conclusion</h3> We show here that DFX is a putative promising drug-sensitizing agent for the treatment of MCL. DFX-co-treatment not only enhances the efficacy of the tested drugs but also restores the anti-proliferative activity of the drugs in resistant MCL cells. To the best of our knowledge, this study is the first to provide evidence on the potential of DFX to overcome drug resistance in MCL.

The Synergistic Anti-Tumor Activity of EZH2 Inhibitor SHR2554 and HDAC Inhibitor Chidamide through ORC1 Reduction of DNA Replication Process in Diffuse Large B Cell Lymphoma.

Simple SummaryThe EZH2-targeted drugs have demonstrated notable therapeutic effects in EZH2 mutant B-cell lymphoma patients. In this study, we demonstrated that the combination of EZH2 inhibitor SHR2554 and HDAC inhibitor HBI8000 exert synergistic anti-proliferative activity in both EZH2 wide-type and mutation B-cell lymphoma. More importantly, gene expression profile analysis revealed simultaneous treatment with these agents led to dramatic inhibition of DNA replication initiator protein ORC1, which might contribute to great efficacy of combination strategy. The combination of EZH2 inhibitor and HDAC inhibitor could provide a potential therapeutic treatment for both EZH2 wide-type and mutation B-cell lymphoma patients.Background: Upregulation of H3K27me3 induced by EZH2 overexpression or somatic heterozygous mutations were implicated in lymphomagenesis. It has been demonstrated that several EZH2-target agents have notable therapeutic effects in EZH2-mutant B-cell lymphoma patients. Here we present a novel highly selective EZH2 inhibitor SHR2554 and possible combination strategy in diffuse large B-cell lymphoma (DLBCL). Methods: Cell proliferation, cell cycle and apoptosis were analyzed by CellTiter-Glo Luminescent Cell Viability Assay and flow cytometry. Western Blot was used to detect the expression of related proteins. The gene expression profiling post combination treatment was analyzed by RNA-Seq. Finally, CDX and PDX models were used to evaluate the synergistic anti-tumor effects of the combination treatment in vivo. Results: The novel EZH2 inhibitor SHR2554 inhibited proliferation and induced G1 phase arrest in EZH2-mutant DLBCL cell lines. The combination of EZH2 inhibitor SHR2554 with histone deacetylase (HDAC) inhibitor chidamide (hereafter referred to as HBI8000) exerted synergistic anti-proliferative activity in vitro and in vivo. Gene expression profile analysis revealed dramatic inhibition of the DNA replication process in combined treatment. Conclusions: SHR2554, a potent, highly selective small molecule inhibitor of EZH2, inhibited EZH2-mutant DLBCL more significantly in vitro and in vivo. The combination of HDAC inhibitor HBI8000 with EZH2 inhibitor SHR2554 exhibited dramatic anti-tumor activity in both mutant and wild-type DLBCL, which may become a potential therapeutic modality for the treatment of DLBCL patients.

Synergistic Antitumor Activity of SH003 and Docetaxel via EGFR Signaling Inhibition in Non-Small Cell Lung Cancer.

Epidermal growth factor receptor (EGFR) is overexpressed in lung cancer patients. Despite treatment with various EGFR tyrosine kinase inhibitors, recurrence and metastasis of lung cancer are inevitable. Docetaxel (DTX) is an effective conventional drug that is used to treat various cancers. Several researchers have studied the use of traditional herbal medicine in combination with docetaxel, to improve lung cancer treatment. SH003, a novel herbal mixture, exerts anticancer effects in different cancer cell types. Here, we aimed to investigate the apoptotic and anticancer effects of SH003 in combination with DTX, in human non-small-cell lung cancer (NSCLC). SH003, with DTX, induced apoptotic cell death, with increased expression of cleaved caspases and cleaved poly (ADP-ribose) polymerase in NSCLC cells. Moreover, SH003 and DTX induced the apoptosis of H460 cells via the suppression of the EGFR and signal transducer and activator of transcription 3 (STAT3) signaling pathways. In H460 tumor xenograft models, the administration of SH003 or docetaxel alone diminished tumor growth, and their combination effectively killed cancer cells, with increased expression of apoptotic markers and decreased expression of p-EGFR and p-STAT3. Collectively, the combination of SH003 and DTX may be a novel anticancer strategy to overcome the challenges that are associated with conventional lung cancer therapy.

An open-label phase II study of dostarlimab (TSR-042), bevacizumab (bev), and niraparib combination in patients (pts) with platinum-resistant ovarian cancer (PROC): cohort A of the OPAL trial

Objectives: Preclinical evidence suggests that PARP inhibition (PARPi), anti-PD-1 therapy, and anti-angiogenic therapies have interactions that may support synergistic antitumor activity in pts with PROC. This phase 2 study evaluated activity of combination therapy with the PARPi niraparib, the PD-1 inhibitor dostarlimab, and bev in pts with PROC. Methods: Eligible pts had high-grade, platinum-resistant (progressed ≤6 mo after completion of ≥4 cycles of platinum-based chemo), recurrent epithelial ovarian, fallopian tube, primary peritoneal cancer, or recurrent carcinosarcoma of the ovary (high-grade mixed histology permitted). Pts had 1–2 prior lines of anticancer therapy for OC, and no prior therapy with an anti-PD-1/-L1 or PARPi. Pts received a regimen of 500 mg dostarlimab Q3W x 4, then 1000 mg Q6W + 15 mg/kg bev Q3W + niraparib 300 mg or 200 mg (for weight <77 kg or platelet count <150,000/µL at screening) QD until discontinuation. The primary endpoint was investigator-assessed objective response rate (ORR) per RECIST v1.1. Secondary objectives were progression-free survival (PFS), safety, and disease control rate (DCR). A posthoc analysis by biomarker (BRCA mutation [BRCAm] status, homologous recombination repair mutation [HRRm], and combined positive score [CPS; a measure of intratumoral and immune infiltrate PD-L1 expression], prior lines of therapy, and prior bev use) was performed. Results: Conclusions: Conclusion: Triplet therapy with niraparib, dostarlimab, and bev is tolerable and demonstrated clinical activity in pts with PROC, most of which were BRCA or HRR wt. AEs were as expected. Clinical trial identification: NCT03574779. Funding: GlaxoSmithKline. Preclinical evidence suggests that PARP inhibition (PARPi), anti-PD-1 therapy, and anti-angiogenic therapies have interactions that may support synergistic antitumor activity in pts with PROC. This phase 2 study evaluated activity of combination therapy with the PARPi niraparib, the PD-1 inhibitor dostarlimab, and bev in pts with PROC. Eligible pts had high-grade, platinum-resistant (progressed ≤6 mo after completion of ≥4 cycles of platinum-based chemo), recurrent epithelial ovarian, fallopian tube, primary peritoneal cancer, or recurrent carcinosarcoma of the ovary (high-grade mixed histology permitted). Pts had 1–2 prior lines of anticancer therapy for OC, and no prior therapy with an anti-PD-1/-L1 or PARPi. Pts received a regimen of 500 mg dostarlimab Q3W x 4, then 1000 mg Q6W + 15 mg/kg bev Q3W + niraparib 300 mg or 200 mg (for weight <77 kg or platelet count <150,000/µL at screening) QD until discontinuation. The primary endpoint was investigator-assessed objective response rate (ORR) per RECIST v1.1. Secondary objectives were progression-free survival (PFS), safety, and disease control rate (DCR). A posthoc analysis by biomarker (BRCA mutation [BRCAm] status, homologous recombination repair mutation [HRRm], and combined positive score [CPS; a measure of intratumoral and immune infiltrate PD-L1 expression], prior lines of therapy, and prior bev use) was performed. Conclusion: Triplet therapy with niraparib, dostarlimab, and bev is tolerable and demonstrated clinical activity in pts with PROC, most of which were BRCA or HRR wt. AEs were as expected.

Self-assembly nanovaccine containing TLR7/8 agonist and STAT3 inhibitor enhances tumor immunotherapy by augmenting tumor-specific immune response

BackgroundCancer vaccines are a promising strategy for cancer immunotherapy. Cancer vaccines elicits a specific cytotoxic immune response to tumor antigens. However, the efficacy of traditional peptide-based cancer vaccines is limited...

Abstract 968: Focal adhesion kinase (FAK) inhibitor APG-2449 sensitizes ovarian tumors to chemotherapy via CD44 downregulation

Abstract Ovarian cancer is one of the deadliest malignancies in women, and up to 70% of patients with epithelial ovarian cancer have FAK overexpression, amplification, or activation. FAK plays an important role in cellular migration, growth factor signaling, cell cycle progression, cellular survival and chemoresistance. This biomarker is also significantly associated with higher tumor stage, metastasis, and shorter overall survival in patients with ovarian cancer. Inhibition of FAK is therefore emerging as a promising treatment target. APG-2449 is a clinical stage FAK/ALK/ROS1 multi-kinase inhibitor. In this study we investigated antitumor activity of APG-2449 combined with standard-of-care chemotherapeutics in ovarian cancer in the preclinical setting. In a mouse xenograft tumor model derived from ovarian cancer cell line OVCAR-3, which was resistant to platinum-based therapies, APG-2449 combined with paclitaxel, and paclitaxel plus carboplatin, synergistically enhanced antitumor activity, whereas the chemotherapeutics showed no activity. Synergistic antitumor activity was also observed in multiple patient-derived xenograft (PDX) models derived from women with chemoinsensitive ovarian cancer, which also frequently expresses high levels of FAK. By comparing gene expression profiles of PDX tumors obtained from responders and nonresponders to the combined therapy, we identified CD44 (a marker for cancer stem cells) as a potentially predictive biomarker. Western blot analysis confirmed higher protein levels of CD44 in pretreated tumors of responders. Interestingly, downregulation of CD44 levels was observed in combination-treated tumors, suggesting that these combinations reduced cancer stem cell populations in ovarian cancer. Accordingly, in ovarian cancer cells exposed to APG-2449 alone or combined with paclitaxel, numbers of cells positive for CD44 or aldehyde dehydrogenase 1 (ALDH1; another marker for cancer stem cells) decreased in a dose-dependent manner. In summary, our data suggest that FAK inhibition by APG-2449 sensitizes ovarian tumors to chemotherapeutics in preclinical tumor models of ovarian cancer. The synergistic antitumor activity was mediated by downregulation of CD44+ or ALDH1+ cancer stem cell populations. These findings encourage clinical development of APG-2449 in combination with chemotherapies for treatment of ovarian cancer. Citation Format: Ran Tao, Douglas D. Fang, Yuanbao Li, Kaixiang Zhang, Chunhua Xu, Xu Fang, Qixin Wang, Dajun Yang, Yifan Zhai. Focal adhesion kinase (FAK) inhibitor APG-2449 sensitizes ovarian tumors to chemotherapy via CD44 downregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 968.

Abstract 1137: PRMT5 inhibition regulates alternative splicing and DNA damage repair pathways in SF3B1 R625G expressing uveal melanoma cells

Abstract PRMT5 (protein arginine methyltransferase 5) is a predominant Type II PRMT that catalyzes symmetric dimethylation of protein arginine residues (sDMA). PRMT5 is overexpressed in many types of cancer and plays roles in multiple essential biological processes to promote cancer growth. Previous studies have shown that PRMT5 is a critical molecule for RNA processing and pre-mRNA splicing. Mechanistically, PRMT5 directly methylates arginine residues of several splicing factors such as Small nuclear ribonucleoprotein (SNRPB and SNRPD3) and Serine and arginine rich splicing factor 1 (SRSF1), which contributes to spliceosome assembly and promotes canonical splicing of many essential genes in cancer cells. In the present study, we examined the effects of PRT543, a potent and selective PRMT5 inhibitor, on alternative splicing in uveal melanoma which frequently express hotspot mutations on Splicing factor 3b subunit 1 (SF3B1). We first confirmed that PRT543-treated MEL202 (SF3B1R625G active mutant) and MEL270 (SF3B1WT) cells show significantly increased global alternative splicing, such as increased retained intron (RI) and skipping exon (SE), determined by delta-PSI (percentage of splice-in) analysis. PRT543 downregulates SF3B1 target genes such as FBXW5, MAP3K7, MBD4 and BRD9 that are associated with increased retention of specific intron sites. Interestingly, downregulation of the SF3B1 target genes are more significant in MEL202 (SF3B1R625G) than MEL270 (SF3B1WT), indicating that PRT543 can regulate the activity of the SF3B1 gain of function mutant. Consistent with previously reported PRMT5 knockout studies in hematological cancer cells, PRT543 also downregulates expression of SRSF1 target genes such as POLD1 and PNKP through increased intron retention in primary and metastatic uveal melanoma cell lines. Furthermore, we uncover that PRT543 strikingly increases retention of a specific intron site of ATM (ex33-34), resulting in a significant reduction of ATM protein levels in PRT543-treated MEL202 and MEL270 cells. Gene set enrichment analysis (GSEA) further reveals that PRT543 significantly and specifically regulates DNA replication and repair pathways in MEL202 cells. Importantly, combining PRT543 with DNA-alkylating agents or PARP inhibitors yields a synergistic reduction in cell viability. In summary, our results suggest that PRMT5 inhibition regulates cancer-associated RNA splicing machinery and the DNA damage response, resulting in synergistic antitumor activity when combined with chemotherapy and/or PARP inhibitors, particularly in cancers with spliceosomal mutations. PRT543 is currently under evaluation in a Phase I clinical trial in patients with advanced solid tumors and hematological malignancies (NCT03886831). Citation Format: Koichi Ito, Venkat Thodima, Jack Carter, Neha Bhagwat, Monisha Sivakumar, Alexander Grego, Joseph Rager, Mizue Terai, Takami Sato, Omar Abdel-Wahab, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. PRMT5 inhibition regulates alternative splicing and DNA damage repair pathways in SF3B1 R625G expressing uveal melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1137.

Abstract 1393: PSMA-Targeted Thorium Conjugate (BAY 2315497) and olaparib combination show synergistic anti-tumor activity in prostate cancer models

Abstract The PSMA targeted thorium-227 conjugate PSMA-TTC (BAY 2315497) is a novel targeted alpha therapy (TAT) approach which is currently under investigation in a FiH trial in patients with metastatic castration resistant prostate cancer (mCRPC; NCT03724747). It consists of the alpha emitter thorium-227 complexed to a 3,2-HOPO chelator conjugated to a PSMA targeting antibody delivering radiation to PSMA expressing tumor cells and their microenvironment resulting in the induction of potentially cytotoxic DNA double-strand breaks (DBS). Combining with the PARP inhibitor olaparib may act synergistically via inhibition of DDR pathways. Here we evaluated the efficacy of PSMA-TTC in combination with olaparib that demonstrated a positive outcome in patients with homologous recombination repair-mutated mCRPC. We explored potential synergies of the combination treatment in prostate cancer models in vitro and in vivo and studied the mode of action by protein and RNA analysis. In vitro, synergy of the combination of PSMA-TTC with olaparib with a combination index (CI) of 0.7 was observed in three cell lines, namely LNCaP, 22Rv1 and MDA-PCa2b. with the first two (LNCaP and 22Rv1) carrying mutations in BRCA2. Only moderate synergy with a CI of 0.8 was seen in BRCA2 wt VCaP cells. In vivo, dose dependent anti-tumor activity with single i.v. injections of PSMA-TTC at 150 and 300 kBq/kg was seen in the LNCaP prostate cancer model resulting in a tumor/control (T/C) ratio of 0.3 and 0.09 at day 28, respectively. In contrast, olaparib given at 50 mg/kg QD did not show activity at day 28. The 300 kBq/kg PSMA-TTC dose combined with olaparib showed the most pronounced anti-tumor activity (T/C=0.04) and significantly increased the number of animals with complete tumor regressions compared to high dose PSMA-TTC alone (6 CRs vs 2 CRs, p = 0.0397, Chi-square) on day 28. At the end of the consecutive surveillance period on day 117, 7/8 (87.5%) animals remained tumor-free (putatively cured) in the combination group vs 4/10 (40%) mice in the PMSA-TTC monotherapy group (Chi-square test, P = 0.0400). Additive or synergistic toxicological effects for the combination treatment were not seen based on body weight loss and fatal tox rate. At the 117-day time point both the 300 kBq/kg PSMA-TTC monotherapy and combination with olaparib group demonstrated 0.3% and 9.3% body weight gain indicating good tolerability. Changes in markers of DNA damage response following treatment will be presented. In summary, combination of PSMA-TTC with olaparib shows synergistic preclinical anti-tumor activity and support further clinical investigation of this combination treatment in prostate cancer patients. Citation Format: Christoph A. Schatz, Stefanie Hammer, Antje Wengner, Urs B. Hagemann, Dominik Mumberg, Arne Scholz. PSMA-Targeted Thorium Conjugate (BAY 2315497) and olaparib combination show synergistic anti-tumor activity in prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1393.

Abstract 58: Potent immunotherapy of human placental CD34+-derived natural killer cells with high affinity and cleavage resistant CD16 (CYNK-101) plus Trastuzumab for HER2+ gastric cancer

Abstract Introduction: Overexpression of human epidermal growth factor receptor 2 (HER2) has been reported in ~20% of gastric cancer cases and correlated with poor outcome. Celularity is developing CYNK-101, an allogeneic, off-the-shelf human placental CD34+-derived NK cell product with genetically modified CD16 variant (CD16VP) for cancer treatment. CYNK-101 is designed to enhance the anti-tumor antibody dependent cellular cytotoxicity (ADCC) activity by expressing high-affinity and cleavage resistant CD16VP. Here we report the preclinical efficacy data of CYNK-101 plus Trastuzumab, an anti-HER2 monoclonal antibody, against HER2+ gastric cancer cells. Methods: Human placental CD34+ cells were transduced with lentivirus expressing CD16VP and cultured with cytokines to generate CYNK-101. ADCC activity of CYNK-101 plus Trastuzumab against HER2+ gastric cancer cells was measured by real-time xCELLigence, and cytokine secretion was quantified by Luminex. Ex vivo-CYNK-101 was isolated from NOD-scid IL2Rγnull immunodeficient (NSG) mice 13 days post CYNK-101 infusion. A subcutaneous NCI-N87 xenograft NSG-hIL15 transgenic mouse model was used for in vivo study. Results and conclusion: CYNK-101 was generated from multiple placental CD34+ donors (n=7) with &amp;gt;90% CD56+CD3-and 74.1 ± 5.6% CD16 expression. While 4h PMAi treatment resulted in &amp;gt;89% CD16 cleavage on non-transduced NK cells, &amp;lt;11% cleavage from CYNK-101 demonstrated CD16 shedding resistance. At an effector to target (E:T) ratio of 1:1, CYNK-101 (n=7) showed enhanced lysis of NCI-N87 cells in the presence of Trastuzumab compared to IgG control, 59.6 ± 13.7% vs. 9.8 ± 3.2% at 4h, 97.1 ± 7.1% vs. 26.2 ± 16.1% at 24h, respectively (p&amp;lt;0.001), and secreted increased GM-CSF, IFN-γ, and TNF-α at 24h. Ex vivo-CYNK-101 plus Trastuzumab exhibited enhanced cytotoxicity against NCI-N87 compared to IgG control (30.9% vs. 6.6% at 4h, 90.7% vs. 9.1% at 24h) at the E:T of 0.5:1, as well as increased production of GM-CSF, IFN-γ, and TNF-α at 24h. Compared to CYNK-101 pre-infusion, ex vivo-CYNK-101 showed not only a higher ADCC activity against NCI-N87, but also a more matured NK phenotype with increased expression of CD16, KIR, NKG2D, and CD94. In addition, CD16 shedding resistance was demonstrated on ex vivo-CYNK-101. CYNK-101 in vivo anti-tumor ADCC activity was assessed in a subcutaneous xenograft NCI-N87 mouse model. Significant tumor reduction was shown in the animals treated with CYNK-101 plus Trastuzumab compared to vehicle control, Trastuzumab or CYNK-101 alone (P&amp;lt;0.0001, n=20 mice per group). In summary, our results demonstrated synergistic anti-tumor ADCC activities of CYNK-101 plus Trastuzumab against HER2+ gastric cancer cells in vitro, ex vivo and in vivo. Further development of CYNK-101 in combination with Trastuzumab for HER2+ gastric cancer immunotherapy is underway. Citation Format: Lin Kang, Shuyang He, Irene Raitman, Salvatore Rotondo, Joseph Gleason, Valentina Rousseva, Xuan Guo, Hemlata Rana, Qian Ye, Robert Hariri, Xiaokui Zhang. Potent immunotherapy of human placental CD34+-derived natural killer cells with high affinity and cleavage resistant CD16 (CYNK-101) plus Trastuzumab for HER2+ gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 58.