Killing Cancer Research Articles

Abstract 55: TT-702, a selective and potent A2B receptor antagonist for the treatment of cancer

Abstract Adenosine signaling via the A2A and A2B receptors is emerging as an important regulatory mechanism of immune responses. While A2AR has high affinity for adenosine, A2BR is a low-affinity receptor that becomes activated only under pathological conditions when adenosine concentration reaches micromolar ranges. Besides high levels of adenosine (up to 100 μM), the tumor microenvironment (TME) is characterized by hypoxia. Hypoxia upregulates A2BR on tumors and on immune cells. TT-702 is a novel, selective and highly potent, orally active A2BR antagonist. Here we show that TT-702 inhibits tumor growth through multiple mechanisms including enhancement of dendritic cell (DC) function and T-cell activation, reduction of MDSCs and Tregs, and suppression of cancer cell growth, fibrosis and angiogenesis. The cellular potency of TT-702 was assessed in NECA induced cAMP production. The ability of TT-702 to reverse NECA-mediated immune suppression was evaluated using standard human DC differentiation and MLR assays. The effects of TT-702 on tumor growth were determined in B16F10 melanoma and MC38 and CT26 colorectal cancers in mice treated with TT-702 at 0.3, 1, 3 or 10 mg/kg (BID), anti-PD-1 (RMP1-14, 5 mg/kg, Q2D) or TT-702+RMP1-14 intraperitoneally. TT-702 binds to human A2BR with Ki value of 8.3 nM and exhibits greater than 100x selectivity for A1, A2A and A3 receptors. It potently inhibited NECA-stimulated cAMP production (IC50=2.8 nM). TT-702 dose-dependently inhibited the growth of human prostate cancer cells LNCaP and PC-3 (IC50=1.4 μM). Combination of TT-702 with enzalutamide showed an additive and synergistic effects to inhibit LNCaP and PC-3 growth, respectively. TT-702 prevented NECA-induced suppression of human DC differentiation and completely reversed NECA-induced T cell inactivation (EC50=0.45 μM). By contrast, preladenant, an A2AR antagonist, had no effect on cancer cell growth and DC differentiation. Moreover, TT-702 increased MC38 tumor-infiltrating immune stimulatory cells (DCs, CD4+ and CD8+ T cells) and reduced immunosuppressive cells (MDSCs and Tregs). TT-702 dose-dependently inhibited the growth of mouse MC38 and CT26 colorectal cancers. Interestingly, TT-702 also inhibited the growth of mouse B16F10 melanoma cold tumor. Furthermore, combination of TT-702 with anti-PD-1 synergistically inhibited both B16F10 and CT26 tumor growth. Most importantly, TT-702 significantly reduced tumor fibrosis and angiogenesis, suggesting that TT-702 may improve the TME for T cell penetration, cancer killing and prevention of metastasis. TT-702, a novel, selective and potent A2B receptor antagonist, has the potential to treat a diverse range of hard-to-treat and aggressive tumors (including cold ones) as a monotherapy, and in combination with anti-PD-1, enzalutamide and other anticancer therapies. TT-702 exhibits excellent safety and PK profiles in animals and is slated to enter clinical development in 2021. Citation Format: Peidong Fan, Filomena Housley, Helena Haberstock, Ken Horne, Jim Liu, Elfatih Elzein, Lina Yao. TT-702, a selective and potent A2B receptor antagonist for the treatment of 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 55.

Abstract 2688: The landscape of precision cancer combination therapy: a single-cell perspective

Abstract The availability of single-cell transcriptomics data opens new opportunities for rational design of combination cancer treatments in a systematic manner. Mining such data, we explore the landscape of optimal combination therapy targets in solid tumors (including brain, head and neck, melanoma, lung, breast and colon cancers). To this end, we developed MadHitter (https://github.com/ruppinlab/madhitter), which analyzes tumor single-cell transcriptomics data using combinatorial algorithms to identify precision combination of treatment targets that are predicted to maximize the killing of cancer cells while minimizing the killing of noncancerous ones. We started with a predefined set of 533 proteins that are lowly abundant across healthy tissues and thus may have low off-tumor targeting and toxicity if targeted via chimeric antigen receptors (CAR) T cell therapy. In most cancer types analyzed, we find that combinations composed of a single-digit number of targets are sufficient to kill at least 80% of the tumor cells while killing at most 10% of the non-tumor cells in each patient. We identify targets that are shared across datasets of the same cancer type (e.g., TNR) or across different cancer types (the SOX family, MAGE family, and KRT16). Next, we expanded our search for a set of 1269 known cell surface receptors, which may be precisely targeted by antibody or nanoparticles delivering a toxin into cells via receptor mediated endocytosis. The resulting optimal target sets are encouragingly also in the single-digit size. The gene PTPRZ1, a tyrosine phosphatase receptor, is frequently found in the optimal combinations for brain and head and neck cancers, and EGFR is a high-coverage target in multiple tumor types. Notably, requiring more stringent levels of cancer killing, the number of targets that need to be combined rises sharply in both search spaces described above. In sum, this analysis provides the first systematic characterization of potential combinatorial targets in solid tumors, uncovering promising future targets for both CAR therapy and conjugated toxin delivering antibodies and nanoparticles. Citation Format: Saba Ahmadi, Pattara Sukprasert, Rahulsimham Vegesna, Sanju Sinha, Natalie Artzi, Samir Khuller, Alejandro A. Schäffer, Eytan Ruppin. The landscape of precision cancer combination therapy: a single-cell perspective [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 2688.

Abstract LB068: A novel CD20/CD3 T cell engager designed from Rituximab targeting CD20+ cancer with multiple mechanisms

Abstract Background Bispecific T cell engaging antibodies (TEAs) with one arm targeting a cancer antigen and another arm binding to CD3 have demonstrated impressive efficacy in multiple clinical studies. However, establishing a balance of safety versus efficacy remains challenging. For instance, some TEAs have severe safety issues such as causing cytokine release symptom. Additionally, due to the heterogeneity of cancer cells, not all patients or all cancer cells of one patient respond equally to TEA treatments. It has been shown that cancer cells with PD-L1 overexpression respond to TEA therapy worse than other cancer cells. To develop next generation bispecific TEAs with a better balance of safety, efficacy and expanded mechanism of actions, we replaced heavy chain complementarity-determining regions (HCDRs) in one arm of Rituximab with HCDRs from a CD3 antibody to make a novel CD20/CD3 bispecific antibody, Rituximab/CD3 (GB261). After several rounds of computer aided sequence optimization, the final lead molecule demonstrated a superior balance of safety and efficacy in both in vitro and animal studies. Methods The affinities of antibodies to antigens were determined using FACS and SPR. T cell activation was determined by measuring CD69+/CD2+ cell percentage using FACS and cytokine release. Cancer cell killing assays were performed by measuring target cell depletion after incubating with PBMCs, ADCC assays were performed by measuring target cell depletion after incubating with NK92-CD16 cells and CDC was performed by measuring target cell depletion after incubating with complements-enriched human serum. In vivo cancer cell killing was performed using human PBMC engrafted NDG mouse models. Pre-tox and PK study were performed with cynomolgus monkey.Results GB261 exhibited high affinity to CD20 and an extremely low affinity to CD3. It showed similar T cell activation and cancer cell killing in Rituximab resistant Raji cells (RRCL) while reducing cytokine secretion compared to a benchmark CD20/CD3 (BM). GB261-induced ADCC and CDC only killed CD20+ cells but not CD3+ cells. When RRCL and PBMC cells (1:1) were co-injected with antibodies, it exhibited cancer killing effect comparable to the BM in a PBMC engrafted mouse model. However, if RRCL cells were injected first, then PBMC cells and antibodies were co-injected 4 days later, it showed superior cancer cell killing. Moreover, it delayed cancer relapse more effectively than Rutuximab in mice. Pre-tox and PK data in a cyno model also suggested that GB261 has good safety and PK. Finally, GB261 also demonstrated excellent manufacturability based on expression level and bio-analytic data.Conclusions GB261 exhibits enhanced efficacy and safety, and great manufacturability, presenting several advantages over the BM antibody. Thus, GB261 is a promising bispecific therapeutic antibody against CD20+ cancers. Citation Format: Wenyan Cai, Jianbo Dong, Sachith Gallolu Kankanamalage, Allison Titong, Jiadong Shi, Zhejun Jia, Bo Wang, Cai Huang, Jing Zhang, Jun Lin, Steven Z. Kan, Joe Zhou, Yue Liu. A novel CD20/CD3 T cell engager designed from Rituximab targeting CD20+ cancer with multiple mechanisms [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 LB068.

Oncolytic parainfluenza virus combines with NK cells to mediate killing of infected and non-infected lung cancer cells within 3D spheroids: role of type I and type III interferon signaling

BackgroundThere is intense interest in developing novel oncolytic viruses, which can be used in cancer therapies along with immune cells such as natural killer (NK) cells. We have previously developed...

Carbon quantum dots/Bi4O5Br2 photocatalyst with enhanced photodynamic therapy: killing of lung cancer (A549) cells in vitro

Carbon quantum dots/Bi4O5Br2 photocatalyst with enhanced photodynamic therapy: killing of lung cancer (A549) cells in vitro

Tanshinone IIA enhances susceptibility of non-small cell lung cancer cells to NK cell-mediated lysis by up-regulating ULBP1 and DR5.

Natural killer (NK) cells have a great potential in cancer immunotherapy. However, their therapeutic efficacy is clinically limited owing to cancer cell immune escape. Therefore, it is urgently necessary to develop novel method to improve the antitumor immunity of NK cells. In the present study, it was found that the natural product tanshinone IIA (TIIA) enhanced NK cell-mediated killing of non-small cell lung cancer (NSCLC) cells. TIIA in combination with adoptive transfer of NK cells synergistically suppressed the tumor growth of NSCLC cells in an immune-incompetent mouse model. Furthermore, TIIA significantly inhibited the tumor growth of Lewis lung cancer (LLC) in an immune-competent syngeneic mouse model, and such inhibitory effect was reversed by the depletion of NK cells. Moreover, TIIA increased expressions of ULBP1 and DR5 in NSCLC cells, and inhibition of DR5 and ULBP1 reduced the enhancement of NK cell-mediated lysis by TIIA. Besides, TIIA increased the levels of p-PERK, ATF4 and CHOP. Knockdown of ATF4 completely reversed the up-regulation of ULBP1 and DR5 by TIIA in all detected NSCLC cells, while knockdown of CHOP only partly reduced these enhanced expressions in small parts of NSCLC cells. These results demonstrated that TIIA could increase the susceptibility of NSCLC cells to NK cell-mediated lysis by up-regulating ULBP1 and DR5, suggesting that TIIA had a promising potential in cancer immunotherapy, especially in NK cell-based cancer immunotherapy.

T Cells Plead for Rejuvenation and Amplification; With the Brain's Neurotransmitters and Neuropeptides We Can Make It Happen.

T cells are essential for eradicating microorganisms and cancer and for tissue repair, have a pro-cognitive role in the brain, and limit Central Nervous System (CNS) inflammation and damage upon injury and infection. However, in aging, chronic infections, acute SARS-CoV-2 infection, cancer, chronic stress, depression and major injury/trauma, T cells are often scarce, exhausted, senescent, impaired/biased and dysfunctional. People with impaired/dysfunctional T cells are at high risk of infections, cancer, other diseases, and eventually mortality, and become multi-level burden on other people, organizations and societies. It is suggested that “Nerve-Driven Immunity” and “Personalized Adoptive Neuro-Immunotherapy” may overcome this problem. Natural Neurotransmitters and Neuropeptides: Glutamate, Dopamine, GnRH-II, CGRP, Neuropeptide Y, Somatostatin and others, bind their well-characterized receptors expressed on the cell surface of naïve/resting T cells and induce multiple direct, beneficial, and therapeutically relevant effects. These Neurotransmitters and Neuropeptides can induce/increase: gene expression, cytokine secretion, integrin-mediated adhesion, chemotactic migration, extravasation, proliferation, and killing of cancer. Moreover, we recently found that some of these Neurotransmitters and Neuropeptides also induce rapid and profound decrease of PD-1 in human T cells. By inducing these beneficial effects in naïve/resting T cells at different times after binding their receptors (i.e. NOT by single effect/mechanism/pathway), these Neurotransmitters and Neuropeptides by themselves can activate, rejuvenate, and improve T cells. “Personalized Adaptive Neuro-Immunotherapy” is a novel method for rejuvenating and improving T cells safely and potently by Neurotransmitters and Neuropeptides, consisting of personalized diagnostic and therapeutic protocols. The patient’s scarce and/or dysfunctional T cells are activated ex vivo once by pre-selected Neurotransmitters and/or Neuropeptides, tested, and re-inoculated to the patient’s body. Neuro-Immunotherapy can be actionable and repeated whenever needed, and allows other treatments. This adoptive Neuro-Immunotherapy calls for testing its safety and efficacy in clinical trials.

Inhibition of HIF-1α accumulation in prostate cancer cells is initiated during early stages of mammalian orthoreovirus infection

Mammalian orthoreovirus (MRV) is a safe and effective cancer killing virus that has completed Phase I-III clinical trials against numerous cancer types. While many patients experience benefit from MRV therapy, pre-defined set points necessary for FDA approval have not been reached. Therefore, additional research into MRV biology and the effect of viral therapy on different tumor genetic subtypes and microenvironments is necessary to identify tumors most amenable to MRV virotherapy. In this work we analyzed the stage of viral infection necessary to inhibit HIF-1α, an aggressive cancer activator induced by hypoxia. We demonstrated that two viral capsid proteins were not necessary and that a step parallel with virus core movement across the endosomal membrane was required for this inhibition. Altogether, this work clarifies the mechanisms of MRV-induced HIF-1α inhibition and provides biological relevance for using MRV to inhibit the devastating effects of tumor hypoxia.

Targeting RECQL5 Functions, by a Small Molecule, Selectively Kills Breast Cancer in Vitro and in Vivo.

Clinical and preclinical data reveal that RECQL5 protein overexpression in breast cancer was strongly correlated with poor prognosis, survival, and therapeutic resistance. In the current investigation, we report design, synthesis, and specificity of a small molecule, 4a, which can preferentially kill RECQL5-expressing breast cancers but not RECQL5 knockout. Our stringent analysis showed that compound 4a specifically sensitizes RECQL5-expressing cancers, while it did not have any effect on other members of DNA RECQL-helicases. Integrated approaches of organic synthesis, biochemical, in silico molecular simulation, knockouts, functional mutation, and rescue experiments showed that 4a potently inhibits RECQL5-helicase activity and stabilizes RECQL5-RAD51 physical interaction, leading to impaired HRR and preferential killing of RECQL5-expressing breast cancer. Moreover, 4a treatment led to the efficient sensitization of cisplatin-resistant breast cancers but not normal mammary epithelial cells. Pharmacologically, compound 4a was orally effective in reducing the growth of RECQL5-expressing breast tumors (human xenograft) in NUDE-mice with no appreciable toxicity to the vital organs.

Iron oxide nanoparticles conjugated curcumin to promote high therapeutic efficacy of curcumin against hepatocellular carcinoma

BackgroundCurcumin’s excellent conjugation capability with iron oxide nanoparticles plays a crucial role in enhancing both properties. Iron oxide, as a nanocarrier, will improve curcumin anticancer activity, while curcumin, as a capping agent, will minimize iron oxide nanoparticles toxicity and optimize their biomedical use. MethodologyCytotoxic and antioxidant effects of free curcumin (free Cur) and curcumin capped iron oxide nanoparticles (IONs@Cur) against human liver cancer cells (HepG2) were investigated in vitro. The genomic effect of free Cur and IONs@Cur on the expression of tumor suppressor gene (TP53), autophagy 4 (ATG-4), and superoxide dismutase (SOD2) genes were assessed using quantitative PCR (qPCR). ResultsIONs@Cur results showed significant cytotoxic effects against HepG2 cells, compared with free Cur, and the measured total antioxidant capacity (TAC) for HepG2 cells treated with IONs@Cur was superior to free Cur. Additionally, the treatment with IONs@Cur significantly upregulated the gene expression levels of SOD2 and TP53. Remarkably, the immunofluorescence staining of ATG-4 demonstrated that curcumin conjugation with IONs potentiated the cancer killing effect of curcumin via inducing autophagy. ConclusionThe promising iron oxide nanocarrier would greatly enhance curcumin anticancer activities against HepG2 cells.

Polymer-Coated Extracellular Vesicles for Selective Codelivery of Chemotherapeutics and siRNA to Cancer Cells.

Combination therapies involving small-interfering RNA (siRNA)-mediated gene silencing and small-molecule drugs are of high interest for cancer treatment. Among the current gene delivery carriers, cell-derived extracellular vesicles (EVs) are particularly promising candidates due to their high biocompatibility, low immunogenicity, in vivo stability, and inherent targeting ability. Here, we developed a multifunctional EV platform capable of selective codelivery of siRNA and doxorubicin (DOX) to cancer cells. siRNA was first loaded into engineered lipid-hybridized EVs (eEVs) to serve as a core. Subsequently, DOX was incorporated into a polyelectrolyte shell surrounding eEVs, which was deposited by layer-by-layer (LbL) assembly. This approach resulted in the production of a stable EV-polymer complex (LbL-eEV) with a diameter of 140.2 ± 9.0 nm and zeta potential of +22.1 ± 0.5 mV. Experiments were performed to assess cellular uptake, cytotoxicity, and gene silencing efficacy in lung adenocarcinoma cells (A549), with noncancerous fibroblast cells (CCL-210) used as a control. The results demonstrated that the LbL-eEV complex can traffic through cells and release siRNA in the cytoplasm, while delivered DOX enters nuclei to induce programmed cell death. Moreover, the inherent selectivity of the particles for cancer cells resulted in effective gene silencing and cancer killing efficiency with reduced cytotoxicity to normal cells. Synchronous delivery of siRNA and DOX was also verified by flow cytometry analysis of single cells. In summary, these data provide a proof of concept for engineering EVs to deliver multiple therapeutics and suggest that LbL-eEVs are a promising drug delivery platform for targeting cancer.

Decidual-Like NK Cell Polarization: From Cancer Killing to Cancer Nurturing.

Natural killer (NK) cells accumulate at the fetal-maternal interface and represent 70% of immune cells in the decidua (dNK) at first-trimester pregnancy; they are immune-tolerant toward the semiallogenic fetus and are "nurturing" and nonkilling NK cells. A subset of NK cells in patients with cancer have features in common with dNK, which include expressing CD56, CD9, CD49a, and CXCR3, being poorly cytotoxic and proangiogenic, and mimicking the decidual nurturing role. In the oncologic patient, several factors can "decidualize" NK cells, turning them into immune-suppressant, growth-promoting proangiogenic cells. Here, we suggest ways to sharpen their blunted blades and intercept and curb their cancer-nurturing attitudes to restore their cytotoxic capabilities.

Carbon Nanodots for On Demand Chemophotothermal Therapy Combination to Elicit Necroptosis: Overcoming Apoptosis Resistance in Breast Cancer Cell Lines.

Simple SummaryCarbon nanodots (CDs) are considered a versatile family of fluorescent, near infrared (NIR) active, and bioeliminable nanoparticles. Accordingly, the CDs application in photothermal therapy and theranostics increased. Problems limiting their use arise from the heterogeneity of most CDs and the lack of exhaustive information on their nanotoxicity at cellular and molecular levels. The lack of these data is often quite dramatic and causes substantial loss of translational value. To overcome this, we developed biocompatible homogenous CDs with a well-known structure as well as efficient red fluorescence and NIR photothermal conversion. The controlled photothermal effect and the on-demand release of the irinotecan successfully kill breast cancer cell lines in absence of relevant cell stress after internalization. We believe that these results provide insights to advance the field with significant impact, paving the way for the design of effective and safe nanomedicines for precision photothermal cancer therapies.Background: Engineered luminescent carbon nanodots (CDs) are appealing nanomaterials for cancer image-guided photothermal therapy combining near infrared (NIR)–triggered hyperthermia, imaging, and drug delivery in a single platform for efficient killing of cancer cells. This approach would allow eliciting synergistic regulated cell death (RCD) routes such as necroptosis, targeting breast cancer cells refractory to apoptosis, thus overcoming drug resistance. Methods: We report the preparation of CDs bearing biotin as a targeting agent (CDs-PEG-BT), which are able to load high amounts of irinotecan (23.7%) to be released in a pulsed on-demand fashion. CDs-PEG-BT have narrow size distribution, stable red luminescence, and high photothermal conversion in the NIR region, allowing imaging of MDA-MB231 and MCF-7 cancer cells and killing them by photothermal and chemotherapeutic insults. Results: Cellular uptake, viability profiles, and RCD gene expression analyses provided insights about the observed biocompatibility of CDs-PEG-BT, indicating that necroptosis can be induced on-demand after the photothermal activation. Besides, photothermal activation of drug-loaded CDs-PEG-BT implies both necroptosis and apoptosis by the TNFα and RIPK1 pathway. Conclusions: The controlled activation of necroptosis and apoptosis by combining phototherapy and on-demand release of irinotecan is the hallmark of efficient anticancer response in refractory breast cancer cell lines in view of precision medicine applications.

The Confounders of Cancer Immunotherapy: Roles of Lifestyle, Metabolic Disorders and Sociological Factors.

Simple SummaryThe human immune system is robustly equipped to keep unnatural cell growth in check in the body to suppress cancer progression. However, the cells and molecules of the immune system responsible for preventing cancer growth are often severely compromised in patients harboring this disease. Therefore, to elicit a functional immune response against cancer, scientists have developed antibodies, known as checkpoint inhibitors (CPI), which unleash the compromised immune cells and potentiate them with cancer killing ability. Although CPI revolutionized the treatment of certain cancers, many patients do not respond to the CPI and the treatment outcome varies disproportionately between cancer types. This review elaborates on how lifestyle, metabolic and sociological factors play a role in determining the outcome of CPI treatment. We also discuss potential ways to enhance CPI efficacy by mitigating the effect of these confounding variables.Checkpoint blockade immunotherapy (CPI) is an effective treatment option for many types of cancers. Irrespective of its wide clinical implications, the overall efficacy remains unpredictable and even poor in certain pathologies such as breast cancer. Thus, it is imperative to understand the role of factors affecting its responsiveness. In this review, we provide an overview on the involvement of sociological factors, lifestyles and metabolic disorders in modulating the CPI response in patients from multiple malignancies. Lifestyle habits including exercise, and diet promoted therapeutic responsiveness while alcohol consumption mitigated the CPI effect by decreasing mutational burden and hampering antigen presentation by dendritic cells. Metabolic disorder such as obesity was recognized to enhance the PD-1 expression while diabetes and hypertension were consequences of CPI therapy rather than causes. Among the sociologic factors, sex and race positively influenced the CPI effectiveness on account of increased effector T cell activity and increased PD-1 expression while ageing impaired CPI responsiveness by decreasing functional T cell and increased toxicity. The combined effect of these factors was observed for obesity and gender, in which obese males had the most significant effect of CPI. Therefore these variables should be carefully considered before treating patients with CPI for optimal treatment outcome.

Tackling Resistance to Cancer Immunotherapy: What Do We Know?

Cancer treatment has evolved tremendously in the last few decades. Immunotherapy has been considered to be the forth pillar in cancer treatment in addition to conventional surgery, radiotherapy, and chemotherapy. Though immunotherapy has resulted in impressive response, it is generally limited to a small subset of patients. Understanding the mechanisms of resistance toward cancer immunotherapy may shed new light to counter that resistance. In this review, we highlighted and summarized two major hurdles (recognition and attack) of cancer elimination by the immune system. The mechanisms of failure of some available immunotherapy strategies were also described. Moreover, the significance role of immune compartment for various established cancer treatments were also elucidated in this review. Then, the mechanisms of combinatorial treatment of various conventional cancer treatment with immunotherapy were discussed. Finally, a strategy to improve immune cancer killing by characterizing cancer immune landscape, then devising treatment based on that cancer immune landscape was put forward.

Abstract 1459: Selective killing of SMARCA4-deficient gynecologic cancers by mitochondria oxidative phosphorylation inhibitors

Abstract Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) and dedifferentiated carcinoma of the ovary or endometrium are rare but highly aggressive types of gynecologic cancers. We and others have recently demonstrated that genetic inactivation of SMARCA4, one of the two ATPases of the SWI/SNF chromatin remodeling complex, along with protein loss is the only recurrent somatic mutation of SCCOHT. Furthermore, SMARCA4 loss appears as a key event in the development of a subset of dedifferentiated carcinoma of the ovary, endometrium or other organs. These SMARCA4-deficient dedifferentiated tumors and SCCOHT usually lose the expression of SMARCA2, the alternative ATPase of the SWI/SNF complex. Through mining the publicly available genome-wide CRISPR screen database from the Broad Institute DepMap Project for about 500 cell lines, we identified that SMARCA4/A2-dual deficient ovarian cell lines are selectively sensitive to the genetic ablation of multiple components of mitochondria electron transfer chain (ETC). Using seahorse metabolism assays, we revealed that SCCOHT cells have reduced glycolysis in comparison to other ovarian cancer cells and utilize mitochondria respiration for energy production regardless of the availability of glucose. Accordingly, both SCCOHT and SMARCA4-deficient dedifferentiated ovarian carcinoma cell lines are remarkably more sensitive to several inhibitors of ETC complex I, and tigecycline, a selective inhibitor of the mitochondria ribosomal translation, than other ovarian cancer cells, such as SMARCA4-intact ovarian high-grade serous carcinoma cells and clear cell ovarian carcinoma cells regardless of the expression level of ARID1A, a SWI/SNF complex subunit frequently lost in clear cell ovarian carcinoma. Re-expression of SMARCA4 increased the expression of SLC2A1, encoding the glucose transporter GLUT1, and decreased the sensitivity of SCCOHT cells to ETC complex I inhibitors, suggesting that SMARCA4 loss may reduce the transport of glucose leading to reduced glycolysis and increased reliance on mitochondria respiration, which is currently under investigation. Therefore, our data suggest that selective targeting mitochondria respiratory complex function can be an effective strategy for SCCOHT and other SMARCA4-deficient dedifferentiated cancers of gynecologic tract or other organs. Citation Format: Yemin Wang, Dionzie Ong, Shary Yuting Chen, Eunice Li, Krystal Orlando, Elizabeth Raupach, Jennifer Ji, Bernard Weissman, Patrick Pirrotte, David Humtsman. Selective killing of SMARCA4-deficient gynecologic cancers by mitochondria oxidative phosphorylation inhibitors [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 1459.

Abstract 3400: Adenosine receptor antagonists exhibit potent and selective off-target killing of FOXA1-high cancers

Abstract Drugs targeting adenosine receptors were originally developed for the treatment of Parkinson's disease and are now being tested in immuno-oncology clinical trials in combination with checkpoint inhibitors. We recently reported the killing activity of 4,518 drugs against 578 diverse cancer cell lines determined using the PRISM molecular barcoding approach. Surprisingly, three established adenosine receptor antagonists (CGS-15943, MRS-1220, and SCH-58261) showed potent and selective killing of FOXA1-high cancer cell lines without the need for immune cells. FOXA1 is a lineage-restricted transcription factor in luminal breast cancer, hepatocellular carcinoma, and prostate cancer without known small molecule inhibitors. We find that cytotoxic activity is limited to adenosine antagonists with a three-member aromatic core bound to a furan group, thus indicating a potential off-target mechanism of action. To identify genomic modulators of drug response, we performed genome-wide CRISPR/Cas9 knockout modifier screens. Killing by CGS-15943 and MRS-1220 was rescued by knockout of the aryl hydrocarbon receptor (AHR) and its nuclear partner ARNT. In confirmatory studies, knockout of AHR completely rescued killing by CGS-15943 in multiple cell types. Co-treatment with an AHR small molecule antagonist also rescued cell viability. Knockout of adenosine receptors did not alter drug response. Given that AHR is a known transcriptional regulator, we performed global mRNA sequencing to assess transcriptional changes induced by CGS-15943. The top two genes induced were the p450 enzymes CYP1A1 and CYP1B1. To determine sufficiency, we overexpressed CYP1A1 in a resistant cell line. Ectopic CYP1A1 expression sensitized to CGS-15943-mediated killing. Mass spectrometry revealed covalent trapping of a reactive metabolite by glutathione and potassium cyanide following in vitro incubation with liver microsomes. In addition, treatment of breast cancer cells with CGS-15943 for 24 hours resulted in increased γ-H2AX phosphorylation by western blot, indicative of DNA double stranded breaks. In summary, we identified off-target anti-cancer activity of multiple established adenosine receptor antagonists mediated by activation of AHR. Future studies will evaluate the functional contribution of FOXA1 and activity in vivo. Starting from a phenotypic screening hit, we leverage functional genomics to unlock the underlying mechanism of action. This project will pave the way for developing more effective therapies for biomarker-selected cancers, with potential to improve the care of patients with liver, breast, and prostate cancer. Citation Format: Steven M. Corsello, Ryan D. Spangler, Ranad Humeidi, Caitlin N. Harrington, Rohith T. Nagari, Ritu Singh, Vickie Wang, Mustafa Kocak, Jordan Rossen, Amael Madec, Nancy Dumont, Todd R. Golub. Adenosine receptor antagonists exhibit potent and selective off-target killing of FOXA1-high cancers [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 3400.

Melanoma Peptide MHC Specific TCR Expressing T-Cell Membrane Camouflaged PLGA Nanoparticles for Treatment of Melanoma Skin Cancer.

Melanoma is one of the most aggressive skin cancers, and the American Cancer Society reports that every hour, one person dies from melanoma. While there are a number of treatments currently available for melanoma (e.g., surgery, chemotherapy, immunotherapy, and radiation therapy), they face several problems including inadequate response rates, high toxicity, severe side effects due to non-specific targeting of anti-cancer drugs, and the development of multidrug resistance during prolonged treatment. To improve chemo-drug therapeutic efficiency and overcome these mentioned limitations, a multifunctional nanoparticle has been developed to effectively target and treat melanoma. Specifically, poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) were coated with a cellular membrane derived from the T cell hybridoma, 19LF6 endowed with a melanoma-specific anti-gp100/HLA-A2 T-cell receptor (TCR) and loaded with an FDA-approved melanoma chemotherapeutic drug Trametinib. T-cell membrane camouflaged Trametinib loaded PLGA NPs displayed high stability, hemo- and cyto-compatibility. They also demonstrated membrane coating dependent drug release profiles with the most sustained release from the NPs proportional with the highest amount of membrane used. 19LF6 membrane-coated NPs produced a threefold increase in cellular uptake toward the melanoma cell line in vitro compared to that of the bare nanoparticle. Moreover, the binding kinetics and cellular uptake of these particles were shown to be membrane/TCR concentration-dependent. The in vitro cancer killing efficiencies of these NPs were significantly higher compared to other NP groups and aligned with binding and uptake characteristics. Particles with the higher membrane content (greater anti-gp100 TCR content) were shown to be more effective when compared to the free drug and negative controls. In vivo biodistribution studies displayed the theragnostic capabilities of these NPs with more than a twofold increase in the tumor retention compared to the uncoated and non-specific membrane coated groups. Based on these studies, these T-cell membrane coated NPs emerge as a potential theragnostic carrier for imaging and therapy applications associated with melanoma.

The novel SOD mimetic GC4419 increases cancer cell killing with sensitization to ionizing radiation while protecting normal cells

While radiotherapy is a widely used treatment for many types of human cancer, problems of radio-resistance and side effects remain. Side effects induced by ionizing radiation (IR) arise primarily from its propensity to trigger inflammation and oxidative stress with damage of normal cells and tissues near the treatment area. The highly potent superoxide dismutase mimetic, GC4419 (Galera Therapeutics), rapidly enters cells and is highly effective in dismutating superoxide (O2•−). We performed studies to assess the potency of GC4419 in cancer killing and radio-sensitization in human lung cancer cells and normal immortalized lung cells. Treatment with GC4419 did not alter the radical generation during IR, primarily hydroxyl radical (.OH); however, it quenched the increased levels of O2•− detected in the cancer cells before and following IR. GC4419 triggered cancer cell death and inhibited cancer cell proliferation with no adverse effect on normal cells. Combination of GC4419 with IR augmented the cytotoxic effects of IR on cancer cells compared to monotherapy, while protecting normal cells from IR-induced cell death. DNA fragmentation and caspase-3 activity assays showed that combination of GC4419 with IR enhances cancer cell apoptosis. Moreover, GC4419 increased IR-induced Bax levels with decreased Bcl-2 and elevated Bax/Bcl-2 ratio following treatment. GC4419 increased TrxR activity in the normal cells but decreased activity in cancer cells, conferring increased cancer cell sensitivity to oxidative stress. In conclusion, GC4419 increases the cytotoxic and pro-apoptotic activity of IR in lung cancer cells while decreasing injury in normal cells.

Designing immunogenic nanotherapeutics for photothermal-triggered immunotherapy involving reprogramming immunosuppression and activating systemic antitumor responses

Low tumor mutational burden and absence of T cells within the tumor sites are typical characteristics of “cold immune tumors” that paralyzes the immune system. The strategy of reversing “cold tumors” to “hot tumors” infiltrated high degree of T cells in order to activate anti-tumor immunity has attracted lots of attentions. Herein, immunogenic core–shell Au@Se NPs is fabricated by gold-selenium coordination bond to realize nanoparticles-mediated local photothermal-triggered immunotherapy. As expected, incorporation of gold nanostars (AuNSs) with improved photothermal stability and conversion efficiency promotes the disintegration and transformation of selenium nanoparticles (SeNPs), thus leading to enhanced cancer cells apoptosis by producing higher hyperthermia. Moreover, the results of in vivo experiments demonstrate that the synergy between SeNPs-mediated chemotherapy and AuNSs-induced photothermal therapy not only generated a localized antitumor-immune response with excellent cancer killing effect under the presence of tumor-associated antigens, but also effectively reprogrammed the tumor associated macrophages (TAMs) from M2 to M1 phenotype with tumoricidal activity to devour distant tumors. Without a doubt, this study not only provides a potent strategy to reverse the immunosuppressive tumor microenvironment, but also offers a new insight for potential clinical application in tumor immunotherapy.