Programmed death ligand 1 (PD L1) expression can reduce the immune response in both infectious diseases and cancers. We thus examined PD L1 expression in cervical intraepithelial neoplasias (CINs) and cancers since they each reflect infection by human papillomavirus (HPV). PD L1 protein was not evident by immunohistochemistry in histologically normal cervical epithelia (0/55) even when adjacent to CIN or cancer. PD L1 expression was much increased in CINs (20/21 = 95%) and cervical squamous cell cancer (56/70 = 80%) and localized to the dysplastic/neoplastic squamous cells and mononuclear cells, respectively. There was also a significant increase (each Po0.001) in PD L1 detection in mononuclear cells when comparing cervical squamous cell cancers to endometrial (22/115 = 19%) and ovarian adenocarcinomas (5/40 = 13%). Co-expression analyses showed that the primary inflammatory cell that contained PD L1 was the CD8+ lymphocyte that strongly concentrated around the dysplastic CIN cells and nests of invasive squamous cancer cells. These data show that PD L1 is a solid biomarker of productive HPV infection of the cervix and that it is significantly upregulated in both the carcinoma and surrounding inflammatory cells in cervical cancer when compared with other gynecologic malignancies. This suggests that anti-PD L1 therapy may have a role in the treatment of cervical cancer.

Multidrug resistance (MDR) is a continuing clinical problem that limits the efficacy of chemotherapy in cancer. The over expression of the ATP-binding cassette (ABC) family G2 (ABCG2) transporter is one of the main mechanisms that mediates MDR in cancer. Molecular modeling data indicated that cariprazine, a dopamine D2/D3 receptor partial agonist, had a significant binding affinity for ABCG2 transporter with a Glide XP score of −6.515. Therefore, in this in vitro study, we determined the effect of cariprazine on MDR resulting from the overexpression of ABCG2 transporters. Alone, cariprazine, at concentrations up to 20 μM, did not significantly decrease cell viability. Cariprazine, at concentrations ranging from 1 to 10 μM, did not significantly alter the cytotoxicity of mitoxantrone (MX) in the parental non-small cell cancer cell line, H460 and colon cancer cell S1. However, cariprazine (1–20 μM) significantly enhanced the efficacy of ABCG2 substrate antineoplastic drug MX in the ABCG2-overexpressing MDR cell line, H460-MX20 and S1M1-80, by reducing the resistance fold from 28 to 1 and from 93 to 1.33, respectively. Cariprazine, in a concentration-dependent (1–20 μM), significantly increased the intracellular accumulation of Rhodamine 123 in S1M1-80. Interestingly, 10 or 20 μM of cariprazine significantly decreased the expression levels of the ABCG2 protein in the colon and lung cancer cell lines, suggesting that cariprazine inhibits both the function and expression of ABCG2 transporters at nontoxic concentrations. Overall, our results suggest that cariprazine, via several distinct mechanisms, can resensitize resistant cancer cells to mitoxantrone.

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“Triple-negative” breast cancer (TNBC), is the most aggressive form of malignant mammary tumors that do not express three cell-surface receptors (estrogen receptor-α, progesterone receptor, and HER2/ERBB3). TNBC accounts for about 15 percent of breast cancer cases, with >42,000 new cases each year and approximately 21,000 deaths. Currently there are no targeted treatments available for TNBC. We found an interesting target associated with TNBC, the mitochondrial fission protein GTPase, dynamin-related protein-1 (Drp1), for which one inhibitor (mdivi-1) is under active investigation. However, mdivi-1 has shown inconsistent antineoplastic activity, low potency and selectivity. This led us to use a 200,000-compound library of commercially available small molecules (Specs), in conjunction with the likely structure of the Drp1-mdivi-1 inhibitory complex, resulting in the identification of twelve thieno-pyrimidin-4-yl-hydrazinylidene class of compounds that were highly potent and selective Drp1 inhibitors. In our preliminary work we found one compound ANT4 with IC50 value of 180-220 nM that has up to 100-fold selectivity in different TNBC cells compared to normal breast cells and up to 30-fold compared to other normal cells. We discovered that ANT4 is a selective inhibitor of Drp1 and has a 100-fold greater affinity for the Drp1 complex. ANT4 inhibited TNBC colony formation, invasion and metastasis,150-fold more potently than Mdivi-1. Interestingly, we discovered that ANT4, in nanomolar range, selectively induced a unique non-apoptotic, necroptotic inducing (NANI) cell death in TNBC cells by increasing the expression of necroptotic cell death markers (RIP, p-RIP, MLKL, p-MLKL). Further, ANT4, inhibited the expression of pro-apoptotic proteins and enhanced the expression of anti-apoptotic proteins. In addition, ANT4 arrested the TNBC cells in S-phase without producing any significant nuclear condensation or release of reactive oxygen species (ROS). Further studies are underway to understand the Drp1 mediated NANI induction process in TNBC cells and to identify the structural requirements that are necessary for targeting cancers refractory to apoptosis. The findings of these studies will allow us to understand the biology of necroptosis-induced Drp1 inhibition and to develop this new class of small molecules for the treatment of TNBC patients. Citation Format: Angelique Nyinawabera, Smiti Gupta, Karthikeyan Chandrabose, Amit K. Tiwari. Necroptosis induction in triple negative breast cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2332.

3524 Background: The activity of pemetrexed is highly dependent on the intracellular enzyme folypolyglutamate synthase (FPGS) which adds glutamates to pemetrexed and yields very potent pemetrexed polyglutamates. Pemetrexed pentaglutamate (tetraglutamated pemetrexed) is 80-fold more potent than pemetrexed in inhibiting thymidylate synthase. Yet it is a poor drug candidate because it cannot readily cross the negatively charged cell membrane due to its own negative charge. We are developing LEAF-1401, a novel nanoliposomal encapsulation of gamma L-pentaglutamated pemetrexed. Because liposomes can readily be taken up by tumor cells, for its anti-tumor effect, LEAF-1401 can directly deliver pentaglutamated pemetrexed into tumor cells, bypassing the need for transmembrane folate carriers and FPGS which are both downregulated in resistant tumors. Methods: To measure drug levels in tumor, blood and various tissues (biodistribution), in vivo testing of LEAF-1401 and pemetrexed was conducted in a CT-26 murine colorectal carcinoma xenograft model. Animals were treated with a single dose of either LEAF-1401 (80mg/kg; equivalent to 32 mg/kg pemetrexed) or pemetrexed (118mg/kg). Tumor growth inhibition and clinical assessments were conducted. Animals were sacrificed: 5 mice per timepoint in each group and tumor, blood, liver, spleen and other tissues were harvested. Pentaglutamated pemetrexed levels were quantitatively analyzed by LC/MS/MS. Results: Compared to pemetrexed, LEAF-1401 treatment resulted in a 19-fold increase in exposure levels of pentaglutamated pemetrexed in the tumor and significant tumor growth inhibition. Plasma levels of pentaglutamated pemetrexed were high with LEAF-1401, but undetectable with pemetrexed. Like other liposomes, LEAF-1401 also resulted in accumulation of pentaglutamated pemetrexed in the liver and spleen (See Table below). Treatment appeared to be generally well tolerated. Conclusions: LEAF-1401, given at approximately a quarter of the equivalent pemetrexed dose, resulted in a 19-fold increase in pentaglutamate pemetrexed in tumor tissue compared to regular pemetrexed. LEAF-1401 represents a promising new class of novel nanoliposomal antifolates, that enhance the intratumoral delivery of potent polyglutamate antifolates, and improve antitumor activity while retaining an acceptable safety profile. [Table: see text]

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Genomic analysis of the human transcriptome has been made possible only in last decade by next generation sequencing (NGS). Recent advancements in NGS has further allowed us to look into small non-coding RNAs (sncRNAs) such as microRNAs (miRNAs), Piwi-interacting-RNAs (piRNAs), long non-coding RNAs (lncRNAs) & small nuclear/nucleolar RNAs (sn/snoRNAs). Recently, the roles of sncRNAs in biological processes have been implicated in biomarker development for diagnosis, prognosis &therapy. In the present study, 50 colon cancer patient’s small RNA sequencing raw data was downloaded from NIH bioproject (PRJNA266667; 7 TNM stage II & 43 TNM stage III) which contained 27 female & 23 male samples. 24 samples had metachronous metastasis (MM) & 26 non-metachronous metastasis (NMM). The small RNA-seq data was analyzed using PartekFlow. In depth analysis showed aberrant expression of 48 miRNAs (all upregulated) in TNM-III vs TNM-II specimens & 20 miRNAs (17 upregulated & 3 downregulated) in MM vs NMM. Further investigation of dysregulated miRNA through pathway analysis confirmed that the majority of the miRNAs were involved in cancer signaling pathways. Analysis of piRNA found unusual expression of 60 piRNAs (57 upregulated & 3 downregulated) in TNM-III vs TNM-II & 31 piRNAs (28 upregulated & 3 downregulated) in MM vs NMM. Further analysis of long non-coding RNAs, we found 77 lncRNAs were significantly expressed in TNM-III vs TNM-II &18 lncRNAs in MM vs NMM. We also, investigated small nuclear/nucleolar RNAs (sn/snoRNAs), miscRNAs & mtRNAs, we identified 37 snRNAs, 105 snoRNAs, 28 miscRNAs & 5 mtRNAs in TNM-III vs TNM-II whereas 2 snRNAs, 11 snoRNAs, 57 miscRNAs & 8 mtRNAs in MM vs NMM were identified. In summary, our comprehensive analysis on publicly available small RNA-seq data identified multiple small non-coding RNAs that need to be further explored for their use in the prognosis, diagnosis & therapy of colon cancer. Citation Format: Srinivas V. Koduru, Angelique Nyinawabera, Dino J. Ravnic, Amit K. Tiwari. Interrogation of small RNA-seq data for small noncoding RNA in human colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3490. doi:10.1158/1538-7445.AM2017-3490

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