Potent and dual monocarboxylate transporter (MCT) 1 and 4 inhibitors have been developed for the first time as potential anticancer agents based on α-cyanocinnamic acid structural template. Candidate inhibitors 1–9 have been evaluated for in vitro cell proliferation against MCT1 and MCT4 expressing cancer cell lines. Potential MCT1 and MCT4 binding interactions of the lead compound 9 have been studied through homology modeling and molecular docking prediction. In vitro effects on extracellular flux via glycolysis and mitochondrial stress tests suggest that candidate compounds 3 and 9 disrupt glycolysis and OxPhos efficiently in MCT1 expressing colorectal adenocarcinoma WiDr and MCT4 expressing triple negative breast cancer MDA-MB-231 cells. Fluorescence microscopy analyses in these cells also indicate that compound 9 is internalized and concentrated near mitochondria. In vivo tumor growth inhibition studies in WiDr and MDA-MB-231 xenograft tumor models in mice indicate that the candidate compound 9 exhibits a significant single agent activity.

Background Breast cancer remains as one of the most lethal types of cancer in women. Among various subtypes, triple-negative breast cancer (TNBC) is the most aggressive and hard to treat type of breast cancer. Mechanistically, increased DNA repair and cell cycle checkpoint activation remain as the foremost reasons behind TNBC tumor resistance to chemotherapy and disease recurrence. Methods We evaluated the mechanism of prexasertib-induced regulation of homologous recombination (HR) proteins using 20S proteasome inhibitors and RT-PCR. HR efficiency and DNA damages were evaluated using Dr-GFP and comet assays. DNA morphology and DNA repair focus studies were analyzed using immunofluorescence. UALCAN portal was used to evaluate the expression of RAD51 and survival probability based on tumor stage, subtype, and race in breast cancer patients. Results Our results show that prexasertib treatment promotes both post-translational and transcriptional mediated regulation of BRCA1 and RAD51 proteins. Additionally, prexasertib-treated TNBC cells revealed over 55% reduction in HR efficiency compared to control cells. Based on these results, we hypothesized that prexasertib treatment induced homologous recombination deficiency (HRD) and thus should synergize with PARP inhibitors (PARPi) in TNBC cells. As predicted, combined treatment of prexasertib and PARPi olaparib increased DNA strand breaks, γH2AX foci, and nuclear disintegration relative to single-agent treatment. Further, the prexasertib and olaparib combination was synergistic in multiple TNBC cell lines, as indicated by combination index (CI) values. Analysis of TCGA data revealed elevated RAD51 expression in breast tumors compared to normal breast tissues, especially in TNBC subtype. Interestingly, there was a discrepancy in RAD51 expression in racial groups, with African-American and Asian breast cancer patients showing elevated RAD51 expression compared to Caucasian breast cancer patients. Consistent with these observations, African-American and Asian TNBC patients show decreased survival. Conclusions Based on these data, RAD51 could be a biomarker for aggressive TNBC and for racial disparity in breast cancer. As positive correlation exists between RAD51 and CHEK1 expression in breast cancer, the in vitro preclinical data presented here provides additional mechanistic insights for further evaluation of the rational combination of prexasertib and olaparib for improved outcomes and reduced racial disparity in TNBC.

Monocarboxylate transporters 1 and 4 (MCT1 and MCT4) are involved in tumor development and progression. Their expression levels are related to clinical disease prognosis. Accordingly, both MCTs are promising drug targets for treatment of a variety of human cancers. The noninvasive imaging of these MCTs in cancers is regarded to be advantageous for assessing MCT‐mediated effects on chemotherapy and radiosensitization using specific MCT inhibitors. Herein, we describe a method for the radiosynthesis of [18F]FACH ((E)‐2‐cyano‐3‐{4‐[(3‐[18F]fluoropropyl)(propyl)amino]‐2‐methoxyphenyl}acrylic acid), as a novel radiolabeled MCT1/4 inhibitor for imaging with PET. A fluorinated analog of α‐cyano‐4‐hydroxycinnamic acid (FACH) was synthesized, and the inhibition of MCT1 and MCT4 was measured via an L‐[14C]lactate uptake assay. Radiolabeling was performed by a two‐step protocol comprising the radiosynthesis of the intermediate (E)/(Z)‐[18F]tert‐Bu‐FACH (tert‐butyl (E)/(Z)‐2‐cyano‐3‐{4‐[(3‐[18F]fluoropropyl)(propyl)amino]‐2‐methoxyphenyl}acrylate) followed by deprotection of the tert‐butyl group. The radiofluorination was successfully implemented using either K[18F]F‐K2.2.2‐carbonate or [18F]TBAF. The final deprotected product [18F]FACH was only obtained when [18F]tert‐Bu‐FACH was formed by the latter procedure. After optimization of the deprotection reaction, [18F]FACH was obtained in high radiochemical yields (39.6 ± 8.3%, end of bombardment (EOB) and radiochemical purity (greater than 98%).

Novel silyl cyanocinnamic acid derivatives have been synthesized and evaluated as potential anticancer agents. In vitro studies reveal that lead derivatives 2a and 2b have enhanced cancer cell proliferation inhibition properties when compared to the parent monocarboxylate transporter (MCT) inhibitor cyano-hydroxycinnamic acid (CHC). Further, candidate compounds exhibit several-fold more potent MCT1 inhibition properties as determined by lactate-uptake studies, and these studies are supported by MCT homology modeling and computational inhibitor-docking studies. In vitro effects on glycolysis and mitochondrial metabolism also illustrate that the lead derivatives 2a and 2b lead to significant effects on both metabolic pathways. In vivo systemic toxicity and efficacy studies in colorectal cancer cell WiDr tumor xenograft demonstrate that candidate compounds are well tolerated and exhibit good single agent anticancer efficacy properties.

A novel MCT1 and MCT4 dual inhibitor reduces mitochondrial metabolism and inhibits tumor growth of feline oral squamous cell carcinoma

Synthesis of highly strained fused substituted dihydrobenzopyran cyclopropyl lactones derived from coumarin carboxylates are reported. The substrate scope tolerates a variety of 6-and 8-substituents on the coumarin ring. Substitution at the 5or 7-position is resistant to tricyclic lactone formation except with 7-methyl substitution. Benzamide-containing coumarins afford the tricyclic ketal. A plausible mechanism is proposed for the formation of the fused lactone: intramolecular rearrangement of trans cyclopropyl methyl ketones with phenolic acetate via the formation of a hemiacetal.

Novel N-phenylindazole based diarylureas have been designed, synthesized and evaluated as potential anticancer agents. In vitro cell viability studies of these derivatives illustrate good potency with IC50 values in the range of 0.4–50 μM in several cancer cell lines including murine metastatic breast cancer 4T1, murine glioblastoma GL261, human triple negative breast cancer MDA-MB-231, human pancreatic cancer MIAPaCa-2, and human colorectal cancer cell line WiDr. The ester group in the lead compound 8i was modified to incorporate amino-amides to increase solubility and stability while retaining biological activity. Further in vitro studies reveal that lead candidates inhibit tube length in HUVEC cells. In vivo systemic toxicity studies indicate that these candidate compounds are well tolerated in mice without any significant side effects. Anticancer efficacy studies in WiDr tumor xenograft and 4T1 tumor syngraft models demonstrate that the lead candidate 11 exhibits significant antitumor properties as a single agent in these tumor models.

Epithelial Ovarian Cancer (EOC) is the fifth leading cause of cancer-related deaths in women in the USA, gets diagnosed at an advanced stage (stage III-IV) with local or distant metastasis. The primary therapeutic regimen of EOC involves surgical removal of all the visible tumor mass followed by platinum-based chemotherapy either as a single agent or in combination with a taxane. Despite initial responses, over 70% of EOC patients develop recurrent disease and they are often resistant to available chemotherapeutic drugs, contributing to poor survival. This appalling situation emphasizes the critical need to identify the molecular mechanisms involving recurrence of these tumors' aggressive growth and developing therapeutic resistance. Our analysis from chemo naïve and recurrent tumors from the same patients' samples, and isogenic ovarian cancer cell lines data showed recurrent tumors and resistant cell lines several upregulated cancer stem cell markers including ALDH1A1, SOX2 and altered expression of DNA damage response and repair genes that are involved in the processing of platinum adducts and crosslink repair. Prominently, RAD6, an E2ubiquitin-conjugating enzyme, is significantly (> 6 folds) overexpressed in recurrent ovarian tumors and associated with aggressive tumor cell growth, stemness, chemoresistance to platinum drugs, and poor prognosis. Our studies validated that, chemotherapy induced upregulated RAD6 reprograms epigenetic milieu in tumor cells through ubiquitination of histone variants H2A, H2AX, and H2B, which further recruits additional epigenetic modifiers to these regions and regulates genes involved in DNArepair, cell survival, stemness, and chemoresistance. Additionally, our mechanistic studies demonstrate RAD6 dependent recruitment of several epigenetic modifiers such as histone methylases and demethylases to regions of ubiquitylated histones and their crosstalk. Collectively, our study presents novel chemotherapy induced epigenetic modulator contributing to therapeutic recurrence of EOC and could be an important therapeutic target to treat chemo-resistant EOC and improve the progression-free survival of patients suffering from this deadly disease. Formatted: Right, Indent: Left: 1" Citation Format: Tasmin Rahman Omy, Shirisha Jonnalagadda, Mark Reedy, Komaraiah Palle. RAD6-mediated epigenetic reprogramming contributes to therapy-induced chemo-resistance in ovarian 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 2111.