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Pancreatic ductal adenocarcinoma (PDAC) displays a dismal prognosis due to late diagnosis and high chemoresistance incidence. For advanced disease stages or patients with comorbidities, treatment options are limited to gemcitabine alone or in combination with other drugs. While gemcitabine resistance has been widely attributed to the levels of one of its targets, RRM1, the molecular consequences of gemcitabine resistance in PDAC remain largely elusive. Here we sought to identify genomic, epigenomic, and transcriptomic events associated with gemcitabine resistance in PDAC and their potential clinical relevance. We found that gemcitabine-resistant cells displayed a coamplification of the adjacent RRM1 and STIM1 genes. Interestingly, RRM1, but not STIM1, was required for gemcitabine resistance, while high STIM1 levels caused an increase in cytosolic calcium concentration. Higher STIM1-dependent calcium influx led to an impaired endoplasmic reticulum stress response and a heightened nuclear factor of activated T-cell activity. Importantly, these findings were confirmed in patient and patient-derived xenograft samples. Taken together, our study uncovers previously unknown biologically relevant molecular properties of gemcitabine-resistant tumors, revealing an undescribed function of STIM1 as a rheostat directing the effects of calcium signaling and controlling epigenetic cell fate determination. It further reveals the potential benefit of targeting STIM1-controlled calcium signaling and its downstream effectors in PDAC.Significance:Gemcitabine-resistant and some naïve tumors coamplify RRM1 and STIM1, which elicit gemcitabine resistance and induce a calcium signaling shift, promoting ER stress resistance and activation of NFAT signaling.

Pediatric brain cancer medulloblastoma standard-of-care results in numerous comorbidities. Medulloblastoma is comprised of four molecular subgroups. Group 3 subgroup patients have the highest relapse rates and 20% survival after standard-of-care. Group 3 tumors have high expression of GABRA5, which codes for the α5 subunit of the γ-aminobutyric acid type A receptor (GABAAR). We are advancing a therapeutic approach for group 3 based on GABAAR modulation using benzodiazepine-derivatives. We performed analysis of GABR and MYC expression in medulloblastoma tumors and used molecular, cell biological, and whole-cell electrophysiology approaches to establish presence of a functional ‘druggable’ GABAAR in group 3 cells. Analysis of expression of 763 medulloblastoma tumors reveals that group 3 tumors share high subgroup-specific and correlative expression of GABR genes, which code for GABAAR subunits α5, β3 and γ2 and 3. There are ~1000 functional α5-GABAARs per group 3 patient-derived cell that mediate a basal chloride-anion efflux of 2x109 ions/sec. Benzodiazepines, designed to prefer α5-GABAAR, impair group 3 cell viability by enhancing chloride-anion efflux with subtle changes in their structure having significant impact on potency. A potent, non-toxic benzodiazepine (‘KRM-II-08’) binds to the α5-GABAAR (0.8 μM EC50) enhancing a chloride-anion efflux that induces mitochondrial membrane depolarization and in response, TP53 upregulation and p53, constitutively phosphorylated at S392, cytoplasmic localization. This correlates with pro-apoptotic Bcl-2-associated death promoter (BAD) protein localization. GABR expression can serve as a diagnostic biomarker for group 3 tumors, while α5-GABAAR is a therapeutic target for benzodiazepine binding, enhancing an ion imbalance that induces apoptosis. Citation Format: Laura Kallay, Havva Keskin, Alexandra Ross, Manali Rupji, Olivia A. Moody, Xin Wang, Guanguan Li, Taukir Ahmed, Farjana Rashid, Michael Rajesh Stephen, Kirsten A. Cottrill, Austin Nuckois, Maxwell Xu, Deborah E. Martinson, Frank Tranghese, Yanxin Pei, James M. Cook, Jeanne Kowalski, Michael D. Taylor, Andrew Jenkins, Daniel Pomeranz Krummel, Soma Sengupta. Modulating native GABAA receptors in medulloblastoma with positive allosteric benzodiazepine-derivatives induces cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2623.

Inhibitors of the proteasome-ubiquitin system have been found to be effective in the clinical management of multiple myeloma. We here describe the compound VLX1570, an optimized lead of the 19S deubiquitinase inhibitor b- AP15. VLX1570 was shown to inhibit the enzymatic activities of both USP14 and UCHL5 in vitro, and was shown to induce accumulation of high molecular weight polyubiquitinated proteins in multiple myeloma cells. The compound showed limited inhibitory activity of a panel of kinases. VLX1570 induces apoptosis and cell death of multiple myeloma cell lines at submicromolar concentrations. Importantly, a multiple myeloma cell line selected for bortezomib resistance was found to be sensitive to VLX1570. VLX1570 was found to be able to overcome apoptosis resistance by these cells. Induction of the phosphorylation of stress-activated kinases (JNK and p38) and inhibition of ERK phosphorylation was observed in multiple myeloma cells. We finally showed that VLX1570 confers antineoplastic activity in two different myeloma xenograft models and conclude that VLX1570 is a candidate agent for treatment of multiple myeloma. Citation Format: Xin Wang, Chitralehka Mohanty, Padraig D'Arcy, Maria Hägg Olofsson, Felicitas Bossler, Asher Chanan-Khan, Stig Linder. Development of the proteasome deubiquitinase inhibitor VLX1570 for treatment of multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4557A. doi:10.1158/1538-7445.AM2015-4557A

As a second-generation nucleoside analogue, clofarabine, is employed primarily for the treatment of paediatric patients with relapsed or refractory acute lymphoblastic leukaemia after at least two prior regimens, but its application toward the solid tumors attracts more and more attention recently. In this study, the effect of clofarabine was investigated in several breast cancer cell lines and compared to a normal cell line (hTERT RPE-1). Upon treatment with 0.1 or 0.2μM clofarabine for 48 hrs, inhibition of cell growth (S-phase arrest) was investigated in both breast cancer and normal cell lines. And we observed that the induction of apoptosis was obvious in the all breast cancer cells but not in the normal cells. More than 40% breast cancer cells entered apoptotic or/and necrotic process after they were treated to 0.2μM clofarabine for 48 hrs. This was associated with an increase in dCK activity in malignant cells between 2.2 and 4.7 folds but only 1.2-fold in hTERT RPE-1 cells. Upon treatment with clofarabine, the cleaved-caspase 3 was observed and the level of anti-apoptotic protein (Bcl-2 and Bcl-xL) down-regulated significantly in the breast cancer cell lines, but not in the hTERT RPE-1 cells. In addition, the sensitivity of all breast cancer cells to the toxicity of clofarabine was attenuated significantly by down-regulation of dCK with siRNA. These results showed that as a single-agent and combination regim clofarabine is worth to be explored in solid tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5674. doi:1538-7445.AM2012-5674

Clofarabine is a new-generation nucleoside analog that was synthesized to combine the most favorable pharmacological properties of its congener fludarabine and cladribine. It acts as a strong inhibitor of DNA polymerase, DNA synthesis and ribonucletide reductase (R1/R2). Like other nucleoside analogues, clofarabine must be activated within cells to the 5′-triphodphate form initially by deoxycytidine kinase (dCK). In this study, the anticancer effect of clofarabine was investigated in three solid tumor cell lines, HCT116 (a colon carcinoma cell line), U1810 (a human non-small cell lung carcinoma cell line), and MCF7 (a mammary carcinoma cell line) and compared to four leukemic cell lines, CCRF-CEM, HL60, K562 and MOLT4. Clofarabine was investigated to enter into HCT116, U1810, and MCF7 in a clearly detectable manner by LigandTracer® White. At 10 µM a high signal is achieved and approaches equilibrium after 1∼2 hrs. Pre-incubation with 10 µM clofarabine resulted in rapid inhibition of thymidine incorporation and DNA synthesis. Induction of apoptosis by clofarabine was cell line dependent, but the average apoptotic level was higher in leukemic cell lines than solid tumor cell lines; however it seemed the sensitivity for apoptosis of cell line is correlated with the activity of dCK and thymidine kinase 1 (TK1) in the solid tumor cell lines. Upon treatment with 10 μM clofarabine for 24 hrs, apoptosis induction increased compared to untreated cells 3.1-, 2.9- and 2.1-fold; the activity of dCK 7.3-, 3.7- and 3.1-fold and the activity of TK1 4.2-, 1.4- and 1.2-fold in U1810, HCT116 and MCF7 cells, respectively. In general, the expression of R1, dCK and TK1 in leukemic cells was higher than in solid tumor cells, but the expression of R2, p53R2 and p53 varied among the cell lines. However, there was an inverse correlation between induction of apoptosis at 100nM clofarabine (upon 24 hours incubation) and the expression of p53R2 (spearman rank test −0.67, p=0.1). In addition, clofarabine treatment causes retardation in S-phase, which seems to be related to the activity of TK1 and the protein expression of R2 in U1810, HCT116 and MCF7 cells. This study is a first step to extend the application of clofarabine toward the solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3654.