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Upper Tract Urothelial Carcinomas (UTUC) are extremely aggressive tumors of ureter or renal pelvis. UTUC present less tumor mutational burden and low tumor immune infiltrate compared to bladder cancer. Despite this they are treated with the same protocol than bladder cancer with more than 50% of relapses justifying the need of new therapeutic options. To improve patient care, we suggest stimulating the immune system by platinum-based chemotherapies (Cisplatin-Gemcitabine (CisGem) or Carboplatin-Gemcitabine (CarboGem)) in order to potentiate the effect of an anti-PD-L1, the Durvalumab. We launched a phase II clinical trial called “iNDUCT” which studies these combinations in UTUC patients. In parallel, we conduct an in vitro project that aims to determine if chemotherapies could transform cold tumor into a hot tumor, and if so by which mechanisms? Using UTUC cell lines (UM-UC-14,UCC03,UCC17,UCC14,UCC47) we have evaluated the cytotoxicity effects of the chemotherapies combinations in 2D and 3D cell cultures. We have assessed their potential (i) to induce DNA damage using image cytometry, (ii) to induce PD-L1 expression using flow cytometry, (iii) to induce immune cell death using ELISA kits, (iv) to activate the cGAS/STING pathway using qPCR and Western-Blot, and finally (v) to attract immune cells by using heterotypic spheroids model (tumoral cells+PBMCs).Our results demonstrate that CisGem and CarboGem present synergistic effects in UTUC spheroid cultures. These treatments also induce DNA damage pathway demonstrated by an increase of γH2AX, P-ATM, P-CHK1 and P-CHK2 positive cells. We found an increase of PD-L1 membrane expression after treatment in UTUC cell lines. RNA Seq analyses indicates that the major pathways upregulated by these combinations are inflammatory pathways (TNF-α signaling via NFkB, interferon alpha response, inflammatory response, interferon gamma response). We could observe an immune cell death induction demonstrated by an increase of ATP and HMGB1 release and calreticulin translocation. We showed cGAS/STING pathway activation as evidenced by an increase of P-IRF3 and interferon stimulated genes (ISGs) expression. We demonstrated an inhibition of the ISGs induction after treatment by our chemotherapies when cells are treated with an ATM inhibitor or in UMUC-14 deleted for STING. And we finally showed that CisGem and CarboGem can increase immune infiltration in the heterotypic tumor spheroids. These results indicate that the combination of platinum salts + gemcitabine induces inflammatory pathways via a non-canonical STING pathway dependent on ATM activation in UTUC model. Furthermore, these combination induce an upregulation of PD-L1 expression and allow immune cells attraction at the tumor. All these data support that a combination CisGem or CarboGem with an anti-PD-L1 will be efficient for UTUC patients. Citation Format: Alexandra Fauvre, Nadia Vie, Mathilde Robin, Clara Taffoni, Nadine Laguette, Julien Faget, Laurent Gros, Aurélie Garcin, Celine Gongora, Nadine Houede. Platinum-based chemotherapy immunomodulatory effects and immunotherapy association in upper tract urothelial carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1294.

Purpose: It has recently been shown that it is possible to improve the prediction of carboplatin clearance by adding plasma cystatin C level (cysC), an endogenous marker of glomerular filtration rate, to the other patient characteristics routinely used for carboplatin individual dosing, namely serum creatinine (Scr), actual body weight (ABW), age, and sex. This multicenter pharmacokinetic study was done to evaluate prospectively the benefit of using cysC for carboplatin individual dosing. Experimental Design: The 357 patients included in the study were receiving carboplatin as part of established protocols. A population pharmacokinetic analysis was done using NONMEM program. Seven covariates studied were as follows: Scr, cysC, age, sex, ABW, ideal body weight, and lean body mass. Results: The best covariate equation was as follows: carboplatin clearance (mL/min) = 117.8. (Scr/75)−0.450. (cysC/1,00)−0.385. (ABW/65)+0.504. (age/56)−0.366. 0.847sex, with Scr in μmol/L, cysC in mg/L, ABW in kilograms, age in years, and sex = 0 for male. Using an alternative weight descriptor (ideal body weight or lean body mass) did not improve the prediction. This final covariate model was validated by bootstrap analysis. The bias (mean percentage error) and imprecision (mean absolute percentage error) were +1% and 15%, respectively, on the total population, and were of a similar magnitude in each of the three subgroups of patients defined according to their body mass index. Conclusion: For the first time, a unique formula is proposed for carboplatin individual dosing to patients, which is shown to be equally valid for underweight, normal weight, and obese patients.

Prostate cancer (PCa) is a major public health issue as it is the second cause of death among cancers in industrialized countries, and its incidence is rising. Surgery is the main option for the curative treatment of localized forms of PCa and recurrences are managed with androgen deprivation strategies. In the case of advanced metastatic PCa and when tumors become resistant to castration (mCRPC), chemotherapy with taxanes is initiated. However, response rates remain low with a restricted number of alternatives due to the intrinsic resistance of CRPC to a wide range of cytotoxic agents. Advanced PCa are particularly resistant to topoisomerase I (Top1) inhibitors from the camptothecin (CPT) family, but the mechanistic bases of this resistance have not been investigated in details. Top1 is a nuclear enzyme that induces transient single strand breaks in duplex DNA to ensure the removal of torsional constraints associated with crucial DNA transactions such as replication, transcription, chromosome segregation or DNA recombination. We have previously shown that interaction of Top1 with DNA-PKcs, a kinase involved in non-homologous end-joining, could regulate the cellular response to CPT independently of DNA repair. This was further confirmed by the fact that NU7441, a specific inhibitor of the kinase activity of DNA-PKcs, had no effect on Hela cell sensitivity to CPT. Recently, it was shown that, in PCa cells, DNA-PKcs could interact with ERG, a transcription factor of the ETS family. As advanced PCa are often characterized by a TMPRSS2-ERG fusion leading to wild-type ERG overexpression, we investigated whether ERG could play a role in the resistance of PCa cells to CPT by regulating Top1/DNA-PKcs interaction. We showed that VCaP cells harboring the TMPRSS2-ERG fusion resulting in a 6-fold increase of ERG protein as compared to ERG fusion-negative (control) DU145 or PC3 cells were >100-fold more resistant to CPT. RWPE-1 stably overexpressing low levels of wild-type ERG (2-fold increase) did not show a major change in resistance to CPT as compared to control RWPE-1 cells stably expressing lacZ. Using two specific siRNA, we also showed that transient downregulation of ERG drastically reduced the sensitivity of VCaP cells to CPT, this effect being dependent on the efficacy of ERG repression. Interestingly, this effect was not due to a change in Top1 levels, suggesting that ERG downregulation may affect Top1-DNA-PKcs interaction. We detected this interaction in VCaP cells by immunoprecipitation in the presence of ethidium bromide, indicating that it was independent of DNA and it remains to be seen whether it is disrupted following ERG repression. Our preliminary findings provide evidences for a new mechanism by which ERG and its interaction with DNA-PKcs could explain, at least in part, the intrinsic resistance of PCa to Top1 inhibitors by the regulation of Top1-DNA-PKcs interaction. Citation Format: Emmanuel Roche, Danièle Montaudon, Samer Kayali, Nadine Houédé, Philippe Pourquier. Role of the ERG transcription factor in the resistance of prostate cancer cells to the topoisomerase I inhibitor camptothecin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3813. doi:10.1158/1538-7445.AM2014-3813

Prostate cancer (PCa) is one of the leading causes of death from cancer in men. High Gleason grade PCa are characterized by aggressive tumors with poorly differentiated cells and a high metastatic potential. They are often refractory to chemical castration but still treated with hormone therapy to which docetaxel or cabazitaxel are added when they become resistant to the anti-androgen. Despite many clinical trials with other chemotherapeutic agents, response rates remain low pointing towards the need for new alternative therapies. Using an in silico approach based on a 86 genes signature which could distinguish between low-grade and high-grade tumors, we identified 9 genes (PCCB, SHMT2, DPM1, RHOT2, RPL13, CD59, EIF4AI, CDKN2C, JUN) for which expression levels across the NCI-60 cell lines panel was significantly correlated (p< 0.05) to oxaliplatin but not to cisplatin sensitivity. Among them, SHMT2 which is overexpressed in high grade PCa, encodes the mitochondrial isoform of serine hydroxymethyl transferase, which converts glycine to serine, which is then exported to the cytoplasm to generate the one-carbon units that are required for the biosynthesis of purines. SiRNA-mediated donwregulation of SHMT2 in DU145 or LNCaP prostate tumor cell lines rendered cells resistant to oxaliplatin but not to cisplatin. CHO 51-11 cells which express a catalytically inactive SHMT2 were also resistant to oxaliplatin but not to cisplatin as compared to CHO K1 control cells. The selective resistance to oxaliplatin was not due to the oxalate moiety since addition of oxalate to cisplatin could not restore the level of sensitivity of CHO cells to oxaliplatin. Selectivity could however be attributed to the diamino cyclohexane (DACH) moiety of oxaliplatin since CHO 51-11 cells were also resistant to dichloro(1,2-diaminocyclohexane)-platinum(II). Resistance to oxaliplatin could also be achieved by SHMT1 downregulation (the cytoplasmic form of SHMT) or by an alteration of the pool of glycine and serine. SHMTs can indirectly regulate the methylation status of DNA, and we showed that siRNA-mediated downregulation of SHMTs in hypomethylated LNCaP cells increased the global level of DNA methylation, as measured by 4 CpGs methylation status of LINE-1. Interestingly, increased level of global methylation status was associated with increased resistance to oxaliplatin. Altogether, our results identified oxaliplatin as a potential alternative for the treatment of high grade prostate cancers and SHMT2 (and SHMT1) as selective markers of oxaliplatin sensitivity which involves, at least in part, the regulation of global levels of DNA methylation. 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 2665. doi:1538-7445.AM2012-2665