Synergistic Tumor Cell Research Articles

NAPRT Silencing in FH-Deficient Renal Cell Carcinoma Confers Therapeutic Vulnerabilities via NAD+ Depletion.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is caused by loss of function mutations in fumarate hydratase (FH) and results in an aggressive subtype of renal cell carcinoma with limited treatment options. Loss of FH leads to accumulation of fumarate, an oncometabolite that disrupts multiple cellular processes and drives tumor progression. High levels of fumarate inhibit alpha ketoglutarate-dependent dioxygenases, including the ten-eleven translocation (TET) enzymes, and can lead to global DNA hypermethylation. Here, we report patterns of hypermethylation in FH-mutant cell lines and tumor samples are associated with the silencing of nicotinate phosphoribosyl transferase (NAPRT), a rate-limiting enzyme in the Preiss-Handler pathway of NAD+ biosynthesis, in a subset of HLRCC cases. NAPRT is hypermethylated at a CpG island in the promoter in cell line models and patient samples, resulting in loss of NAPRT expression. We find that FH-deficient RCC models with loss of NAPRT expression, as well as other oncometabolite-producing cancer models that silence NAPRT, are extremely sensitive to nicotinamide phosphoribosyl transferase inhibitors (NAMPTi). NAPRT silencing was also associated with synergistic tumor cell killing with PARP inhibitors and NAMPTis, which was associated with effects on PAR-mediated DNA repair. Overall, our findings indicate that NAPRT silencing can be targeted in oncometabolite-producing cancers and elucidates how oncometabolite-associated hypermethylation can impact diverse cellular processes and lead to therapeutically relevant vulnerabilities in cancer cells. Implications: NAPRT is a novel biomarker for targeting NAD+ metabolism in FH-deficient HLRCCs with NAMPTis alone and targeting DNA repair processes with the combination of NAMPTis and PARP inhibitors.

Abstract 3305: Golcadomide (CC-99282) is a novel CELMoD® agent with antiproliferative activity and combinatorial potential in disease models of chronic lymphocytic leukemia

Abstract Chronic Lymphocytic Leukemia (CLL) is an adult leukemia characterized by the accumulation of incompetent B lymphocytes expressing CD19, CD20, CD23, and CD5. Current therapies include chemotherapy and combination with targeted therapies such as ibrutinib (Bruton’s tyrosine kinase inhibitor), venetoclax (BCL2 inhibitor), and obinutuzumab (anti-CD20). The tumor microenvironment promotes CLL development and drug resistance. Golcadomide (GOLCA, CC-99282) is an oral cereblon E3 ligase modulator (CELMoD®) agent with immunomodulatory and tumor cell-autonomous activities under clinical investigation for R/R non-Hodgkin lymphomas and CLL/SLL. Here we investigated the effect of GOLCA on inhibiting proliferation and inducing apoptosis of CLL preclinical models by degrading Ikaros and Aiolos. We also studied its combination with other anti-CLL agents. A panel of 10 CLL cell lines and 15 primary CLL patient samples were used to assess efficacy of GOLCA alone or in combination with ibrutinib (Ibru), venetoclax (Ven), and obinutuzumab (Obi). Primary CLL cells were grown ex vivo in a co-culture system with CD40L expressing fibroblasts designed to mimic the CLL lymph node microenvironment. Substrate degradation, viability, cell cycle and immunophenotyping were assessed by flow cytometry. GOLCA showed potent in vitro antiproliferative activity on 6 out of 10 CLL cell lines, including those with high-risk features, with IC50 of 1-20 nM. It also inhibited CLL stimulated cell proliferation and induced apoptosis in all evaluated patient samples in the co-culture system, with low to sub-nanomolar IC50, independent of IGHV mutation status and other chromosomal characteristics. Cell-cycle analysis confirmed inhibition of proliferation in the 5 patient samples evaluated after 3 days of treatment with GOLCA, with a dose-dependent decrease in the fraction of cells in the S phase. GOLCA also demonstrated degradation of the proximal substrates Ikaros and Aiolos in tumor and T cells from CLL patient samples. In combination with Obi, Ven, and Ibru, GOLCA led to synergistic or additive tumor cell toxicity in most patient samples evaluated, suggesting potential clinical benefit of these targeted agent combinations. Additive tumor toxicity was observed in two out of four patient samples when GOLCA was combined with Ibru. We have previously demonstrated that GOLCA induces immune activation in T cells from healthy donor PBMCs. In co-cultures of CLL PBMCs and CD40L-expressing fibroblasts, GOLCA increased T cell numbers and activation characteristics. Ven inhibited this immune activation, while Obi or Ibru maintained the immune activation. Collectively, these findings suggest that GOLCA, alone or in combination with targeted agents, may produce clinical benefit in CLL patients. Citation Format: Antonia Lopez-Girona, Maria Dolores Jimenez-Nunez, Diego Sobradillo, Soraya Carrancio, Preethi Janardhanan, Gauri Deb, Lynda Groocock, Daniel Pierce, Neil Bence, Mark Rolfe. Golcadomide (CC-99282) is a novel CELMoD® agent with antiproliferative activity and combinatorial potential in disease models of chronic lymphocytic leukemia [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 3305.

Abstract 3370: Anti-tumor activity of Debio 0123 in combination with sacituzumab govitecan in preclinical models of breast cancer

Abstract Background: Debio 0123 is a WEE1 inhibitor that has previously shown significant preclinical efficacy in combination with DNA damaging agents and is currently being investigated in phase 1 clinical studies either as a monotherapy or in combination with different standard therapies. Sacituzumab govitecan (SG) is a TROP2 directed antibody-drug-conjugate (ADC) carrying an SN38 payload that has recently gained approval in TNBC and ER+ve breast cancer after demonstrating improvements in survival versus standard therapies. Despite these promising data some patients still do not respond to SG treatment, develop resistance or experience toxic side effects. Herein we present preclinical data that demonstrates Debio 0123 improves tumor response to SG in breast cancer models and provides sustained complete responses and anti-metastatic activity. Methods: Synergy between Debio 0123 and SG was evaluated in breast cancer cell lines, with varying levels of TROP2 expression, in vitro using standard cytotoxicity assay in a matrix combination format. Efficacy of Debio 0123 and SG as monotherapies or in combination was further assessed in vivo, in both TROP2high and TROP2low subcutaneous breast cancer xenograft models at different doses. Results: In vitro, Debio 0123 in combination with SG demonstrated synergistic tumor cell killing in all 4 breast cancer cell lines tested, at all concentrations. In vivo, TROP2high MDA-MB-468 breast cancer xenograft tumors treated with Debio 0123 at 30mg/kg QD resulted in minimal anti-tumor activity as a monotherapy but completely regressed 100% of the tumors in combination with SG. Furthermore, 40% of these complete regressions were sustained for up to 100 days post-treatment initiation. These data were then further replicated at a reduced dose of SG. In the TROP2low MDA-MB-231luc model, Debio 0123 significantly improved response to SG resulting in a 2-fold improvement in tumor growth inhibition. Additionally, combination treatment significantly inhibited metastasis of these tumors to the lung and lymph nodes as monitored by bioluminescence. Collectively, these results lead to a significant improvement in animal survival as a combination compared to either treatment as a monotherapy. Conclusion: These results demonstrate that Debio 0123 synergizes with, and significantly improves breast cancer tumor cell response to, SG regardless of TROP2 expression levels (both in vitro and in vivo), leading to complete regressions and inhibition of metastasis. Collectively these data support Debio 0123 in combination with SG could be a potential therapeutic approach for improving responses in patients with breast cancer. Citation Format: Luke Piggott, Esteban Rodrigo Imedio, Francesco Staehli. Anti-tumor activity of Debio 0123 in combination with sacituzumab govitecan in preclinical models of breast cancer [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 3370.

Abstract A43: Targeting ERK1/2 overcomes resistance to venetoclax by altering Drp1-dependent mitochondrial fission and metabolism in AML

Abstract Background: RAS/RAF/ERK pathway activation is associated with primary or secondary resistance to venetoclax in AML. Oncogenic RAS activates mitochondrial fission through ERK1/2 mediated phosphorylation of the mitochondrial fission GTPase Drp1. Although the deletion of Drp1 inhibits KRAS driven pancreatic cancer, the role of RAS-driven mitochondrial dynamics in AML is unclear. Inhibiting ERK1/2 may, therefore, shed light on AML cells' dependence on RAS driven mitochondrial dynamics and metabolism. Objectives: We assessed the anti-leukemia efficacy of concomitant Bcl2 and ERK1/2 inhibition in preclinical models of venetoclax resistance and further investigated the role of ERK1/2/Drp1-dependent mitochondrial fission in mediating resistance to venetoclax through altered metabolism. Results: ERK1/2 inhibition using Compound 27 (Heightman et al., 2018), sensitized intrinsically resistant and NRAS-mutant OCI-AML3 cells to venetoclax with combination index (CI) of 0.008. Synergy was also confirmed in OCI-AML2 cells with acquired resistance to venetoclax (75% apoptosis versus 22% by venetoclax, CI=0.6). Bulk and CD34+CD38- leukemia initiating cells (LICs) were also eliminated in primary AML samples by the combination (CI:0.03-0.23). Colony formation assay and CyTOF using NRAS-mutant PDX showed impaired clonogenic potential (p=0.0002) and decreased expression of cMyc and CD44 in LICs in response to ERK1/2 inhibition and combination treatment, suggesting impact on stemness. ERK1/2 inhibition using ASTX029 (Heightman et al., 2021) alone and in combination with venetoclax reduced leukemia burden in-vivo and extended survival (p<0.05). Mitochondrial ROS mitigation, Drp1-mediated mitochondrial fission and glycolysis are critical to maintain stemness in AML. ERK1/2 inhibition decreased pDrp1-Ser616 levels along with an increase in mitochondrial length (p<0.001) and mitochondrial ROS (p<0.01), suggesting impaired mitochondrial fission. In OCI-AML3 cells, combination treatment caused downregulation of Drp1 mRNA and decreased phosphorylation of Drp1 at Ser616. Overexpression of a phospho-mimetic i.e. Drp1-Ser616Glu-Ser637Ala in OCI-AML3 cells decreased apoptosis and reversed synergy between ERK1/2 and Bcl-2 inhibition as compared to wild type and phospho-null (Ser616Ala) Drp1 (p<0.001). Of note, overexpression of Drp1-phospho-mimetic led to decreased mitochondrial length, suggesting enhanced fission with a distinct metabolic phenotype including reduced basal respiration, significantly decreased maximal oxygen consumption rate, ATP production and ROS production. Moreover, combination treatment failed to significantly induce ROS production in cells expressing phospho-mimetic Drp1 as opposed to those expressing phospho-null or wildtype Drp1. Conclusion: Inhibition of ERK1/2/Drp1dependent mitochondrial fission and metabolic alterations may be a potential mechanism of synergistic tumor cell killing for the combination treatment. In summary, these data support combining ERK1/2 and Bcl-2 inhibitors in the treatment of AML and warrant further clinical investigation. Citation Format: Priyanka Sharma, Lauren Ostermann, Sujan Piya, Natalia Baran, Anudishi Tyagi, Anna Zal, Christopher Hindley, Kim-Hien Dao, Martin Sims, Tomasz Zal, Vivian Ruvolo, Michael Andreeff, Gautam Borthakur. Targeting ERK1/2 overcomes resistance to venetoclax by altering Drp1-dependent mitochondrial fission and metabolism in AML [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A43.

ERK1/2 Inhibition Overcomes Resistance to Venetoclax in AML By Altering Drp1-Dependent Mitochondrial Fission and Metabolism

Background: Mutational (RAS, PTPN11 etc.) or non-mutational activation of RAS/RAF/ERK pathway is associated with primary or secondary resistance to venetoclax in AML therapy (Zhang et al., Sig Transduct Target Ther. 2022). While upregulation of alternate antiapoptotic proteins e.g. Mcl-1 has been implicated in resistance to venetoclax (Zhang et al., Sig Transduct Target Ther. 2022) role of mitochondrial dynamics (fission/fusion) in venetoclax resistance has not been well explored. Oncogenic RAS activates downstream ERK1/2 phosphorylation and mitochondrial fission through ERK1/2-mediated phosphorylation of the dynamin-like mitochondrial fission GTPase Drp1 (Nagdas et al., Cell Rep. 2019). Drp1 deletion inhibits tumorigenesis of KRAS driven pancreatic cancer, but the importance of RAS driven changes in mitochondrial dynamics in AML is poorly defined. ERK1/2 inhibition may therefore provide insights into the dependence of AML cells on altered mitochondrial dynamics and metabolism driven by oncogenic RAS. Objectives: We assessed the synergistic anti-leukemia efficacy of concomitant Bcl2 and ERK1/2 inhibition in preclinical models of venetoclax resistance and in primary patient samples (n=8). Further, we investigated the role of ERK1/2/Drp1-dependent mitochondrial fission in mediating resistance to venetoclax through altered metabolism. Results: ERK1/2 inhibition using Compound 27 (Heightman et al., J Med Chem. 2018) a close analog of ASTX029, which is currently under clinical investigation in solid tumors (NCT03520075), was highly synergistic with venetoclax at inducing apoptosis in AML cells. NRAS-mutant OCI-AML3 and THP1 cells, that are intrinsically resistant to venetoclax, were sensitized to venetoclax by ERK1/2 inhibition with a combination index (CI) of 0.008 (cell death ~90% for combination versus 20% with venetoclax alone). Synergy was also confirmed in OCI-AML2 cells with acquired resistance to venetoclax, resulting in 75% apoptosis versus 22% by venetoclax alone (CI=0.6). The combination was also effective in eliminating bulk and CD34+CD38- leukemia initiating cells (LICs) in primary AML samples (CI:0.03-0.23). Colony formation assay and single cell level CyTOF analysis using an NRAS-mutant PDX showed markedly impaired clonogenic potential (p=0.0002) and decreased expression of cMyc and CD44 in LICs in response to single agent ERK1/2 inhibition and combination treatment, indicating a potential impact on stemness. Mitochondrial reactive oxygen species (ROS) mitigation, Drp1-mediated mitochondrial fission and glycolysis are critical to maintain stemness in AML (Schimmer et al., Cell Stem Cell. 2018). As a result of ERK1/2 inhibition, pDrp1 (Ser616) levels decreased, and mitochondrial length (p<0.001) and mitochondrial ROS levels (p<0.01) were increased, suggesting impaired fission. In OCI-AML3 cells, combination treatment caused downregulation of Drp1 mRNA and decreased phosphorylation of Drp1 at Ser616. Rescue with overexpression of active phospho-mimetic form of Drp1 i.e. Drp1-Ser616Glu-Ser637Ala in OCI-AML3 cells resulted in decreased apoptosis and reversed the synergy between ERK1/2 and Bcl-2 inhibition when compared to wild type and phospho-null (Ser616Ala) Drp1 overexpression (p<0.001, Fig 1a). Of note, overexpression of Drp1-Ser616Glu-Ser637Ala phospho-mimetic led to decreased mitochondrial length (Fig 1b), suggesting enhanced fission with a distinct metabolic phenotype including reduced basal respiration, decreased maximal oxygen consumption rate (p<0.05) and ATP production (p<0.05), as well as reduced ROS production (p<0.01). Moreover, combination treatment failed to significantly induce ROS production in cells expressing the phospho-mimetic Drp1 as opposed to those expressing phospho-null or wildtype Drp1. Collectively, these results indicate that inhibition of ERK1/2/Drp1dependent mitochondrial fission and metabolic alterations may be a potential mechanism of synergistic tumor cell killing for the combination treatment. Conclusion: ERK1/2 inhibition alters Drp1 dependent mitochondrial dynamics and metabolism, resulting in mitochondrial defects and overcoming resistance to venetoclax by causing apoptosis in venetoclax resistant AML cells. These data provide a strong rationale for the combination of ERK1/2 and Bcl-2 inhibitors in treatment of AML and warrant further investigation in the clinic. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Therapeutic Advancements in Metal and Metal Oxide Nanoparticle-Based Radiosensitization for Head and Neck Cancer Therapy.

Simple SummaryThough radiation therapy remains a primary modality for head and neck cancer (HNC) management, the collateral damage to normal surrounding tissues and tumor relapse represent major challenges. Hence, it is imperative to develop safer and more effective HNC therapies. Metal and metal oxide nanoparticle-based radiosensitizers have been explored for their potential to overcome these challenges. The key impetus of this review was to shed light on ongoing metal and metal oxide nanoparticle-based radiosensitizers’ development and to address their success in in vitro and in vivo HNC models and in clinical translation.Although radiation therapy (RT) is one of the mainstays of head and neck cancer (HNC) treatment, innovative approaches are needed to further improve treatment outcomes. A significant challenge has been to design delivery strategies that focus high doses of radiation on the tumor tissue while minimizing damage to surrounding structures. In the last decade, there has been increasing interest in harnessing high atomic number materials (Z-elements) as nanoparticle radiosensitizers that can also be specifically directed to the tumor bed. Metallic nanoparticles typically display chemical inertness in cellular and subcellular systems but serve as significant radioenhancers for synergistic tumor cell killing in the presence of ionizing radiation. In this review, we discuss the current research and therapeutic efficacy of metal nanoparticle (MNP)-based radiosensitizers, specifically in the treatment of HNC with an emphasis on gold- (AuNPs), gadolinium- (AGdIX), and silver- (Ag) based nanoparticles together with the metallic oxide-based hafnium (Hf), zinc (ZnO) and iron (SPION) nanoparticles. Both in vitro and in vivo systems for different ionizing radiations including photons and protons were reviewed. Finally, the current status of preclinical and clinical studies using MNP-enhanced radiation therapy is discussed.

CHI3L1 regulates PD-L1 and anti-CHI3L1-PD-1 antibody elicits synergistic antitumor responses.

Evasion of the immune response is a hallmark of cancer, and programmed cell death 1 (PD-1) and PD-1 ligand 1 (PD-L1) are major mediators of this immunosuppression. Chitinase 3-like 1 (CHI3L1) is induced in many cancers, where it portends a poor prognosis and contributes to tumor metastasis and spread. However, the mechanism(s) that CHI3L1 uses in metastasis have not been defined. Here we demonstrate that CHI3L1 regulates the expression of PD-L1, PD-L2, PD-1, LAG3, and TIM3 and plays a critical role in melanoma progression and lymphatic spread. CHI3L1 also contributed to IFN-γ-stimulated macrophage PD-L1 expression, and RIG-like helicase innate immunity suppressed CHI3L1, PD-L1, and melanoma progression. Individual antibodies against CHI3L1 or PD-1 had discrete antitumor effects and additive antitumor responses in metastasis models and T cell-tumor cell cocultures when administered simultaneously. Synergistic cytotoxic tumor cell death was seen in T cell-tumor cell cocultures, and significantly enhanced antitumor responses were seen in in vivo tumor models treated with bispecific antibodies that simultaneously target CHI3L1 and PD-1. CHI3L1 contributes to tumor progression by stimulating the PD-1/PD-L1 axis and other checkpoint molecules. The simultaneous targeting of CHI3L1 and the PD-1/PD-L1 axis with individual and, more powerfully, with bispecific antibodies represents a promising therapy for pulmonary metastasis and progression.

Loss of ATRX confers DNA repair defects and PARP inhibitor sensitivity

Alpha Thalassemia/Mental Retardation Syndrome X-Linked (ATRX) is mutated frequently in gliomas and represents a potential target for cancer therapies. ATRX is known to function as a histone chaperone that helps incorporate histone variant, H3.3, into the genome. Studies have implicated ATRX in key DNA damage response (DDR) pathways but a distinct role in DNA repair has yet to be fully elucidated. To further investigate the function of ATRX in the DDR, we created isogenic wild-type (WT) and ATRX knockout (KO) model cell lines using CRISPR-based gene targeting. These studies revealed that loss of ATRX confers sensitivity to poly(ADP)-ribose polymerase (PARP) inhibitors, which was linked to an increase in replication stress, as detected by increased activation of the ataxia telangiectasia and Rad3-related (ATR) signaling axis. ATRX mutations frequently co-occur with mutations in isocitrate dehydrogenase-1 and -2 (IDH1/2), and the latter mutations also induce HR defects and PARP inhibitor sensitivity. We found that the magnitude of PARP inhibitor sensitivity was equal in the context of each mutation alone, although no further sensitization was observed in combination, suggesting an epistatic interaction. Finally, we observed enhanced synergistic tumor cell killing in ATRX KO cells with ATR and PARP inhibition, which is commonly seen in HR-defective cells. Taken together, these data reveal that ATRX may be used as a molecular marker for DDR defects and PARP inhibitor sensitivity, independent of IDH1/2 mutations. These data highlight the important role of common glioma-associated mutations in the regulation of DDR, and novel avenues for molecularly guided therapeutic intervention.

Tumor-Targeting and Penetrating Biomimetic Mesoporous Polydopamine Nanoparticles Manipulate Photo-Thermal Killing and Autophagy Blocking for Synergized Tumor Ablation

How to synergistically manipulate autophagy blocking with significant tumor targeting and penetration for the reinforcement of cancer cell killing during photo-thermal therapy (PTT) remains a substantial challenge. Herein, we fabricated a biomimetic nano-platform by exactly coating homologous cancer cell membranes (CMs) onto the surface of the assembly mesoporous polydopamine nanoparticles (mPDA NPs) with autophagy inhibitor chloroquine (CQ) encapsulating, for synergistically anticancer treatment manipulating PTT and autophagy. The resulting biomimetic mPDA@CMs NPs-CQ system could escape from the macrophage phagocytosis, overcome the vascular barrier, and home in the homologous tumor with high tumor targeting and penetrating efficiency. mPDA NPs core endow the mPDA@CMs NPs-CQ with excellent photo-thermal capability to mediate PTT killing of cancer cells, while NIR-triggered CQ release from mPDA@CMs NPs-CQ could further arrest PTT-induced protective autophagy of cancer cells, thus weakening the resistance of cancer cells to PTT. This PTT killing and autophagy blocking combined anticancer strategy could synergistically induce autophagosome accumulation, ROS generation, mitochondria damage, endoplasmic reticulum stress and apoptotic signal transduction, which leads to synergistic tumor cell inhibition both the in vitro and in vivo. This biomimetic nano-system with synergistically enhanced anticancer efficiency by manipulating PTT killing and autophagy blocking is expected to serve as more effective anticancer strategy.

Ultrasmall Alloy Nanozyme for Ultrasound- and Near-Infrared Light-Promoted Tumor Ablation.

The therapeutic effect of chemodynamic therapy (CDT) is significantly restricted by the stern reaction conditions and slow reaction rate of the Fenton reaction (pH 3-4). Herein, we report an ultrasmall trimetallic (Pd, Cu, and Fe) alloy nanozyme (PCF-a NEs) possessing dynamic active-site synergism, thus exhibiting a cascade glutathione peroxidase and peroxidase (POD) mimicking activities in circumneutral pH. PCF-a NEs exhibit photothermally augmented POD property and high photothermal conversion efficiency (62%) for synergistic tumor cell apoptosis. In addition, ultrasound can also enhance the mass transfer at active catalytic sites of PCF-a NEs, in turn accelerating Fenton-like reaction for tumor-specific CDT. This work provides a strategy for engineering alloy nanozymes in a bioinspired way for the amplification of intratumor reactive oxygen species in response to external stimuli, demonstrating enhanced efficiency for the inhibition of tumor growth in vitro and in vivo.

Tumor-selective, antigen-independent delivery of a pH sensitive peptide-topoisomerase inhibitor conjugate suppresses tumor growth without systemic toxicity.

Topoisomerase inhibitors are potent DNA damaging agents which are widely used in oncology, and they demonstrate robust synergistic tumor cell killing in combination with DNA repair inhibitors, including poly(ADP)-ribose polymerase (PARP) inhibitors. However, their use has been severely limited by the inability to achieve a favorable therapeutic index due to severe systemic toxicities. Antibody-drug conjugates address this issue via antigen-dependent targeting and delivery of their payloads, but this approach requires specific antigens and yet still suffers from off-target toxicities. There is a high unmet need for a more universal tumor targeting technology to broaden the application of cytotoxic payloads. Acidification of the extracellular milieu arises from metabolic adaptions associated with the Warburg effect in cancer. Here we report the development of a pH-sensitive peptide-drug conjugate to deliver the topoisomerase inhibitor, exatecan, selectively to tumors in an antigen-independent manner. Using this approach, we demonstrate potent in vivo cytotoxicity, complete suppression of tumor growth across multiple human tumor models, and synergistic interactions with a PARP inhibitor. These data highlight the identification of a peptide-topoisomerase inhibitor conjugate for cancer therapy that provides a high therapeutic index, and is applicable to all types of human solid tumors in an antigen-independent manner.

Lung cancer chemotherapy using nanoparticles: Enhanced target ability of redox-responsive and pH-sensitive cisplatin prodrug and paclitaxel

Platinum-based combination therapy is more effective and less toxic, but lack of targeting, and is not capable to enrich in the tumor zone. To obstacle these drawbacks, prodrug and nanotechnology strategies have been investigated in this study. GSH-responsive and pH-responsive cisplatin prodrug was synthesized. Cisplatin prodrug and paclitaxel co-loaded nanoparticles: DDP-P/PTX NPs were constructed. The drug release behavior and cytotoxicity of nanoparticles was assessed in vitro. In vivo anticancer efficiency and toxicity were evaluated on lung cancer bearing mice animal model. DDP-P/PTX NPs had a nanoscale size of 112.9 ± 3.5 nm. A reduction and pH triggered drug release with a synergistic tumor cell inhibition ability was observed by DDP-P/PTX NPs. DDP-P/PTX NPs also exhibited high tumor distribution, low systemic toxicity and remarkable antitumor effects in vivo. DDP-P/PTX NPs could be applied as promising anticancer system for the treatment of NSCLC.

A thiol‐bound drug reservoir enhances APR‐246‐induced mutant p53 tumor cell death

The tumor suppressor gene TP53 is the most frequently mutated gene in cancer. The compound APR‐246 (PRIMA‐1Met/Eprenetapopt) is converted to methylene quinuclidinone (MQ) that targets mutant p53 protein and perturbs cellular antioxidant balance. APR‐246 is currently tested in a phase III clinical trial in myelodysplastic syndrome (MDS). By in vitro, ex vivo, and in vivo models, we show that combined treatment with APR‐246 and inhibitors of efflux pump MRP1/ABCC1 results in synergistic tumor cell death, which is more pronounced in TP53 mutant cells. This is associated with altered cellular thiol status and increased intracellular glutathione‐conjugated MQ (GS‐MQ). Due to the reversibility of MQ conjugation, GS‐MQ forms an intracellular drug reservoir that increases availability of MQ for targeting mutant p53. Our study shows that redox homeostasis is a critical determinant of the response to mutant p53‐targeted cancer therapy.

Abstract 569: Rational combination approaches with CRM-1 inhibitor in ovarian cancer

Abstract Background: After the frontline treatment, the majority of patients diagnosed with ovarian cancer at an advanced stage will develop recurrence (80%); thus, there is an urgent need for novel therapeutic approaches. Purpose: Selinexor is a CRM1 inhibitor, which is a selective inhibitor of nuclear export and has demonstrated antitumor effects in ovarian cancer. Here, we examined the efficacy of selinexor alone and in combination with PARP inhibitors in ovarian cancer. As Selinexor reduces the expression of DNA damage repair proteins, we hypothesized that this combination should reduce the number of DNA damage repair proteins, resulting in synergistic tumor cell death. Methods: Western blot was performed to assess baseline CRM1 expression in eleven ovarian cancer cell lines. We tested the efficacy of selinexor alone and in combination with olaparib in ovarian cancer cells (A2780ip2, OVCAR5). Combination Indices (CI) were calculated with CompuSyn and were considered synergistic when CI less than 1 for classic MTT arrays. We used the annexin V-FITC/PI assay to assess apoptosis as a result of selinexor and olaparib alone or in combination. Results: Baseline CRM1 expression was positive in eight of eleven ovarian cancer cell lines tested. The average IC50 in A2780ip2 for selinexor and olaparib was 0.09uM and 0.97uM, respectively. The median combination index was 0.81 in A2780ip2 and 0.92 in OVCAR5 cells. Flow cytometry demonstrated an additive effect of selinexor and olaparib in A2780ip2 cell apoptosis (7.34% and 7.93%, respectively, v. 17.86%). Conclusions: Selinexor demonstrates synergistic activity with olaparib, and this combination warrants further investigation. Citation Format: Katelyn F. Handley, Cristian Rodriguez-Aguayo, Shaolin Ma, Mark S. Kim, Prahlad T. Ram, Anil K. Sood. Rational combination approaches with CRM-1 inhibitor in ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 569.

Abstract 4042: Potent synergistic tumor cell suppression from combination of ONC201 and epigenetic modulators EZH2 or HDAC inhibitors provides a novel treatment strategy for solid tumors

Abstract ONC201 is a promising anti-cancer agent that upregulates cytotoxic TRAIL pathway signaling in cancer cells. It has broad spectrum anti-tumor activity and achieved tumor regression and prolonged disease stabilization in clinical trials, especially in patients with H3K27M mutant gliomas. Based on the fact that the H3K27M mutation reduces the level of H3K27 dimethylation (H3K27me2) and trimethylation (H3K27me3), we hypothesized that cancer cells could be sensitized to ONC201 by epigenetic modulators that impact on histone methylation or acetylation. We tested the synergy between epigenetic modulators and ONC201 in tumor cell lines. We treated cancer cells from different tissue origins including breast cancer, pancreatic cancer, colorectal cancer, GBM, and DIPG with single agent ONC201, EZH2 inhibitor EPZ-6438 or the combination of ONC201 plus EPZ-6438. We also treated cancer cell lines originating from colorectal cancer, breast cancer, gastric cancer, prostate cancer, GBM and DIPG with combination of histone deacetylase inhibitor (HADCi), vorinostat, and ONC201. Cell viability was determined with the Cell Titer-Glo assay. Apoptosis was evaluated through flow cytometry analysis of cell distribution. Integrated stress response (ISR) activity widely observed in ONC201-treated tumor cells was evaluated using Western blot analysis of ATF4 and apoptosis by analysis of PARP cleavage. Cytotoxic TRAIL pathway signaling was evaluated using Western blot analysis of DR5. Our results demonstrate that ONC201 synergistically reduced cell viability, induced integrated stress response, cytotoxic TRAIL pathway signaling and apoptosis in combination with epigenetic modulators. There was greater induction of ATF4 and DR5 in combination therapy-treated tumor cells versus monotherapy. Immunoblotting analysis showed that combination of ONC201 and EZH2 inhibitor EPZ-6438 reduced H3K27me3 and EZH2. ONC201 is an antagonist of dopamine receptor D2 (DRD2). Dopamine receptor D5 (DRD5) is a dopamine receptor family member that opposes DRD2 signaling. The DRD2+DRD5- biomarker signature is associated with enhanced ONC201 tumor cell sensitivity. In order to investigate the role of dopamine receptors in anti-tumor effect of ONC201, we also tested the mRNA level of dopamine receptors by RT-PCR and observed that both vorinostat and EPZ-6438 upregulated DRD5 more than DRD2. Thus, we suspect other target genes may be involved in sensitization to ONC201 due to epigenetic modulation. We are currently investigating the interplay between H3K27 methylation versus acetylation in ONC201 sensitivity with or without additional therapeutic modulation by epigenetic drugs. Our results unravel potent synergy between ONC201 and EZH2 inhibitors or HDAC inhibitors and provide further insights into the role of H3K27me3 in ONC201 drug sensitivity. Citation Format: Yiqun Zhang, Lanlan Zhou, Michael Glantz, Howard Safran, Attila Seyhan, Wafik S. El-Deiry. Potent synergistic tumor cell suppression from combination of ONC201 and epigenetic modulators EZH2 or HDAC inhibitors provides a novel treatment strategy for solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4042.

MYD88 Triggered SYK Activation Promotes BCR Cross-Talk, and Identifies SYK As a Novel Therapeutic Target of Mutated MYD88 Signaling

Activating mutations in MYD88, a component of the Toll-receptor (TLR) pathway, trigger Myddosome self-assembly and multiple downstream pro-survival signaling through BTK and IRAK4/IRAK1 (Yang et al, Blood 2013). Activation of B-cell receptor (BCR) signaling has also been observed in WM, though the mechanism(s) remain to be clarified (Argyropoulos et al, Leukemia 2016). While activating mutations in the BCR components CD79A/B are common in MYD88 mutated ABC DLBCL, they are uncommon in WM (Ngo et al, Nature 2011; Hunter et al, Blood 2014). We therefore sought to clarify if TLR/MYD88 crosstalk could explain aberrant BCR signaling in WM. We performed PhosFlow analysis of MYD88 and BCR signaling components of MYD88 mutated and wild-type WM and ABC DLBCL cell lines. These studies showed high levels for expression of the BCR component p-SYK (Y525-526) in MYD88 mutated WM (BCWM.1, MWCL-1) and ABC DLBCL (TMD-8, HBL-1) cell lines versus MYD88 wild-type cell lines. High levels of p-SYK were also observed in primary MYD88 mutated WM cells compared to healthy donor peripheral blood B-cells. Following treatment with a MYD88 blocking peptide, p-SYK was robustly reduced in WM cell lines, while only modest reduction was observed in ABC DLBCL cell lines which also carry activating CD79B mutations. Use of MYD88 inhibitor also blocked the p-SYK in primary MYD88 mutated WM cells. Lentiviral over-expression of MYD88 L265P but not wild-type MYD88 or vector control augmented p-SYK expression in MYD88 mutated BCWM.1 WM cells, as well as MYD88 wild-type Ramos and OCI-Ly7 cells. Conversely, knockdown of MYD88 in BCWM.1 cells confirmed the dependence of p-SYK on mutated MYD88. p-SYK was also confirmed to be in complex with the Myddosome in BCWM.1 cells by co-immunoprecipitation (co-IP) using anti-MYD88 and anti-SYK/anti-p-SYK specific antibodies. Over-expression and knockdown studies of mutated MYD88 also showed an association of downstream p-STAT3 (Y705) signaling on MYD88 triggered p-SYK, a finding confirmed by use of SYK inhibitors which blocked p-STAT3 in a dose-dependent manner. CellTiter-Glo® viability assays showed the SYK inhibitors, R406 and Entospletinib (GS-9973), produced higher cytotoxicity in MYD88 mutated versus wild-type B-cell lines. (Figure 1A) Since BCR/SYK kinase triggers divergent signaling from TLR/BTK/IRAK pathways, we examined the combined effect of BTK and SYK inhibition. Combination index and normalized isobologram analysis demonstrated synergistic effects with the combination of ibrutinib with either R406 or Entospletinib in MYD88 mutated WM and ABC DLBCL cell lines (Figure 1B, 1C). The combination of ibrutinib with either R406 or Entospletinib also produced more robust tumor cell apoptosis in primary MYD88 mutated WM cells. Our findings show that MYD88 can cross-talk with BCR pathway through SYK tyrosine kinase, and trigger aberrant p-STAT3 signaling. The inhibition of both TLR/BTK and BCR/SYK activation by use of ibrutinib and SYK inhibitors produced synergistic tumor cell killing of MYD88 mutated WM and ABC DLBCL lymphomas, and provides a framework for clinical studies aimed at extinguishing aberrant MYD88 signaling. [Display omitted] DisclosuresCastillo:Millennium: Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Genentech: Consultancy; Beigene: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding. Hunter:Pharmacyclics: Consultancy. Gray:Syros, Soltego, Petra, C4 Therapeutics: Equity Ownership. Treon:Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding; BMS: Research Funding; Johnson & Johnson: Consultancy; Janssen: Consultancy, Other: Travel, Accommodations, Expenses.

Molecular Targeting and Inactivation of Glutaminase and CDK4/6 in Mantle Cell Lymphoma

Mantle Cell Lymphoma (MCL) is an aggressive B‐cell non‐Hodgkin's lymphoma (NHL) subtype that accounts for 7% of all NHL cases. Upon relapse, standard salvage regimens are ineffective and the disease is considered clinically incurable. Hence, novel therapeutic strategies that effectively target deregulated pathways that contribute to MCL pathogenesis are warranted. The hallmark of MCL is a chromosomal translocation t (11:14) event that results in the constitutive expression of the G1‐phase cell cycle regulatory oncogene, cyclin D1 (CCND1), resulting in uncontrolled cell proliferation. Cyclin D1 binds to and activates cyclin dependent kinases 4 and 6 (CDK4/6) and the complex phosphorylates the tumor suppressor gene product retinoblastoma (RB), a negative regulator of the cell cycle. MCL patients whose tumors express cyclin D1 have reduced survival. Uncontrolled cell proliferation results in metabolic alterations of tumor cells to support the energy and biosynthetic needs of unregulated cell growth. Adaptations by malignant cells involve changes in the expression of genes involved in metabolism. This results in both increased glucose and glutamine uptake and metabolism in tumor cells. Recent studies in metabolically reprogrammed tumor cells have shown increased levels of glutaminase, the enzyme involved in the first steps of glutamine metabolism. We hypothesize that molecular targeting of proliferation and glutamine metabolism will induce synergistic tumor cell death. In vitro studies with a MCL cell line were completed using Palbociclib, a potent and selective inhibitor of CDK4/6 which was recently approved by the FDA for the treatment of HR+/HER2− metastatic breast cancer. MTT assays were also carried out using BPTES which inhibits enzymatic activity by binding to the glutaminase tetramer, stabilizing it in an inactive conformation. Cell viability assays show that treatment of MCL cells with Palbociclib resulted in a dose dependent reduction in cell viability. Treatment with 5μM BPTES alone resulted in a slight reduction in cell viability. However, treatment with 5μM BPTES sensitizes cells to treatment with different concentrations of Palbociclib. Studies are ongoing using different concentrations of BPTES with a series of concentrations of Palbociclib to determine the synergistic effects of the combination treatment. As studies continue, quantification of synergy will be relayed using the Chou‐Talalay method with computerized simulation. Preliminary cell cycle assays using flow cytometry show that treatment of cells with Palbociclib induce G1‐phase cell cycle arrest. Establishing a treatment regimen that achieves a synergistic therapeutic effect may allow for integration of a novel molecular targeted approach to treating MCL.Support or Funding InformationButler University Holcomb research award, AFPE Gateway to Research awardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Combination of selective polo-like kinase 1 (PLK1) inhibitor PCM-075 with abiraterone in prostate cancer and non-androgen-driven cancer models.

369 Background: The majority of metastatic castration-resistant prostate cancer (mCRPC) patients will become resistant to abiraterone. Drug combinations with abiraterone offer the possibility to extend patient benefit to anti-androgen therapy. PLK1, an essential mitotic kinase for onset of G2/M transition and cytokinesis, is over-expressed in prostate cancer, correlated positively with Gleason grade and PLK1 RNAi induces G2/M arrest and apoptosis in prostate cancer cells. PCM-075 (formerly NMS-1286937) is a potent, highly selective PLK1 inhibitor currently in clinical trials for the treatment of acute myeloid leukemia. Combination of PCM-075 with abiraterone was examined in mCRPC models and other cancer types for synergistic interaction. Methods: We used a variety of tumor cell lines (C4-2, LNCaP, BT-20, AU565, SK-OV-3), animal and computational modeling techniques to elucidate the mechanism and assess the potential clinical utility of synergistic tumor cell killing by PLK1 inhibitors (PLK1i), including PCM-075, with abiraterone. Results: Synergy was observed between abiraterone and PLK1i, including PCM-075, in models of CRPC (C4-2). In addition, PLK1i and abiraterone were synergistic in murine C4-2 xenografts. Unexpectedly, PLK1i sensitized a subset of non-prostate cancer cells to abiraterone, including AR-negative breast and ovarian cancer cells. In contrast to PLK1i, other mitotic arrest agents do not synergize with abiraterone. To elucidate the molecular basis this synergy, we screened a panel of cell lines and implemented a new computational method that links RNA expression patterns to drug combination synergy. This analysis implicated signaling through the retinoic acid (RA) nuclear receptors as a component of this synergy, which was validated experimentally. Increased basal expression of genes involved in RA metabolism in cancer cells is associated with increased synergy. Conclusions: Abiraterone has AR-independent effects that provide a mechanism and biomarkers predictive for synergistic killing when combined with the PLK1i PCM-075. This combination has potential to extend mCRPC patient benefit to abiraterone regardless of AR status.

Combining ultrasound and intratumoral administration of doxorubicin-loaded microspheres to enhance tumor cell killing

Melanoma is an incurable disease for which alternative treatments to chemotherapy alone are sought. Here, using a melanoma model, we investigated the antitumor potential of combining ultrasound (US) with poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with doxorubicin (DOX). The aim was to achieve synergistic tumoricidal activity through direct and indirect US-mediated damage of tumor cells combined with sustained and potentially controllable release (when combined with US) of DOX from microspheres. An in vitro release assay demonstrated an ability of US to affect the release kinetics of DOX from DOX-loaded PLGA microspheres by inducing a 12% increase in the rate of release. In vitro viability assays demonstrated that combining US with DOX-loaded PLGA microspheres resulted in synergistic tumor cell (B16-F10 melanoma cells) killing. Melanoma-bearing mice were treated intratumorally with DOX (8 µg)-loaded microspheres and subjected to US treatment at the tumor site. This treatment could significantly extend survival (mean survival (MS) = 22.1 days) compared to untreated mice (MS = 10.4 days) and most other treatments, such as blank microspheres plus US (MS = 11.5 days) and DOX (8 µg)-loaded microspheres alone (MS = 13 days). The findings that immune checkpoint blockade did not significantly extend survival of mice treated with DOX (8 µg)-loaded microspheres plus US, and that tumor-free (“cured”) mice were not protected from subsequent tumor rechallenge suggests minimal involvement of the adaptive immune response in the observed antitumor activity. Nevertheless, the synergistic increase in survival of melanoma-challenged mice treated with the combination of US and DOX-loaded microspheres implicates such a treatment methodology as a promising additional tool for combatting otherwise currently incurable cancers.

Abstract IA17: Pathway dependence and drug synergism in NF1-associated malignant peripheral nerve sheath tumors using the zebrafish model

Abstract Malignant peripheral nerve sheath tumors (MPNSTs) are very aggressive and often metastatic soft tissue sarcomas, which are frequently found in patients who have neurofibromatosis type 1 (NF1). Currently, surgical excision is the only curative therapy for MPNST; however, many patients have unresectable or metastatic tumors at diagnosis and the recurrence rate after surgery is high. Chemotherapy regimens are only partially effective and associated with significant toxicity that can severely reduce quality of life. To establish a preclinical model of this highly malignant tumor, we developed a faithful in vivo MPNST transplantation assay to assess pathway dependence. mCherry-labeled MPNSTs cells were harvested from nf1 and p53 mutant zebrafish and implanted into the pericardial space of embryos at 2 dpf. 24 hours later, we added multiple pathway inhibitors or DMSO vehicle to the fish water and the embryos were incubated for 4 days. Drugs in clinical use for MPNST were tested such as inhibitors of MEK (trametinib), RTK (sunitinib), mTOR (rapamycin), HSP90 (ganetespib), microtubules (vincristine), alkylating agents (cytoxan), XPO I (selinexor), topo II (etoposide), and topo I (topotecan). Of these, only topotecan and selinexor showed significant cytotoxic responses at their MTDs. These two inhibitors were also active in human MPNST cell lines, and we are testing to see if their tumor cell cytotoxic effects are synergistic. Clinical trials of Topo I and XPO I inhibitors have not been reported for MPNST, and our results indicate that these two drugs should be evaluated clinically in patients with this tumor. We are now assessing FDA-approved drugs to identify those that synergize with topotecan and selinexor to induce sustained MPNST cell death and improved survival at tolerable dosages. Our long-range goal is to define active combinations of three or more non-cross resistant drugs that exhibit synergistic and sustained tumor cell cytotoxicity at dosages tolerable to the host. Citation Format: Dong Hyuk Ki, Shuning He, A. Thomas Look. Pathway dependence and drug synergism in NF1-associated malignant peripheral nerve sheath tumors using the zebrafish model [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr IA17.