BACKGROUND RESULTS Pancreatic cancer is the fourth leading cause of cancer-related death in developed countries. Although new standard first line regimens, such as FOLFIRINOX and gemcitabine combined with nab-paclitaxel, have improved overall survival, the prognosis of this disease is still very poor with 5-year survival rate of 8%. Thus new treatment options in pancreatic cancer represent a critical medical need (Taieb J et al, Annals Oncology, 2017). Recently, several pre-clinical and clinical studies showed that new therapeutic strategies based on rational combination of drugs could solve the need to improve therapeutic index. Thus, new drugs are required to enhance chemotherapy activity, by modulating the drug targets, metabolism or transport mechanisms. Epigenetic alterations play an important role in initiation and progression of several cancers, including pancreatic cancer. Moreover, in contrast to DNA mutations the “epimutations” must be actively maintained through DNA replication for their dynamic nature, thus being their functional effects reversible and, consequently, targetable. In this prospective, Histone Deacetylase inhibitors (HDACi) are an emerging group of agents that targeting histone and non-histone proteins deacetylation can modulate gene expression as well as cellular functions, regulating different altered pathway in cancer, such as apoptosis, cell cycle and DNA repair (Budillon et al, RecPatAnticanDrugDiscov,2007). The novel HDACi 4SC-202 (domatinostat), is an orally available potent HDACi specific for class I HDAC isoenzymes. It has shown substantial anti-tumour activity in a broad panel of cancer cell lines as well as in various cancer xenograft models. A Phase I study (TOPAS study- NCT01344707) with 4SC-202 in patients with advanced hematological malignancies is currently ongoing (Gruber W et al, Int J Cancer. 2018 - von Tresckow B et al, EurJof Haematology. 2018). Notably, 4SC-202 has been already demonstrated induce a significant decrease stem-cell like characteristics in pancreatic cancer cell lines, evaluating as clonogenic growth (Mishra VK et al, Nucleic Acids Res. 2017). In order to improve therapeutic efficacy of conventional treatment and overcome drug resistance, we test potential combination strategies of 4SC- 202 plus chemotherapy (CT) regimens such as gemcitabine/paclitaxel (GP) or 5’DFUR/SN38 (capecitabine and irinotecan metabolite, respectively) (CAPIRI), in pancreatic cancer models. Moreover, we investigate 4SC-202 effects on cancer stem cells (CSCs), a cellular subpopulation which drives tumorigenic metastatic properties of the entire cancer, as well as carries on resistance to chemotherapy and radiotherapy. 4sc-202 (domatinostat), a novel histone deacetylase inhibitor, improves chemotherapy efficacy and overcomes drug resistance in pancreatic cancer models. Maria Serena Roca 1 , Alessandra Leone 1 , Simona Di Rienzo 1 Tania Moccia 1 , Elena Di Gennaro 1 and Alfredo Budillon 1 1 Experimental Pharmacology Unit, Istituto Nazionale Tumori Fondazione G. Pascale - IRCCS, 80131 Naples, Italy SIC Conference 2018 September 19 th -22 th , Milan, Italy KEY FINDINGS Synergistic antitumor effects of 4SC-202 plus Chemotherapeutics in a simultaneous and sequential schedules of treatment Cell lines Histology Tumor source Mutational status PANC1 Adenocarcinoma Primary Tumour CDKN2A KRAS TP53 ASPC1 Adenocarcinoma metastasis, ascites CDKN2A FBXW7 KRAS MAP2K4 TP53 A Figure 1. Histone deacetylase inhibitor in pancreatic cancer cell lines. (A) The histology, tumor source and genetic background of the three cell lines that recapitulate the heterogeneity of PDAC Pancreatic cancer cell lines features and 4sc-202 molecular characteristics SRB Assay after 96h SRB Assay after 72h E SRB Assay after 96h SRB Assay after 72h A Figure 3. Synergistic Effect of 4SC-202 plus gemcitabine/paclitaxel (GP). Standard care treatment is to date represented by chemotherapy, either alone or with radiation therapy (CTR), after surgery. However, despite clinical improves by different schedules, combining several chemothepautic drugs (i.e. gemcitabine plus paclitaxel or fluorouracil plus irinotecan and oxaliplatin), most, if not all, patients after an initial response eventually relapse as a result of acquired drug resistance (Taieb J et al, Ann of Oncology. 2017). We explored the anti-proliferative and cytotoxic effects of 4SC-202 plus gemcitabine/paclitaxel or 5’DFUR/SN38 (capecitabine and irinotecan metabolite, respectively) (CAPIRI), in PANC1 (blue), AspC1 (green) and PANC28 (red) cancer cell lines, evaluating a simultaneous and sequential schedules of treatment. (A) In the upper table, the combination 4SC-202 plus gemcitabine, as single drug, showed synergism in PANC28 cell line, upon simultaneous exposure to equitoxic doses • 4SC-202 (domatinostat), a new epigenetic modulator, inhibits pancreatic tumor growth by an antiproliferative effect and by preventing clonogenic formation. Moreover, 4SC-202 blocks cell cycle in G2 phase and induces apoptosis and necrosis. • Combined treatment with equipotent doses 4SC-202 and standard chemotherapy, such as GP (Gemcitabine plus Paclitaxel) and CAPIRI (5’DFUR and Irinotecan) resulted in synergistic/additive anti-proliferative effect in three pancreatic cancer cell lines. Interestingly, an increase of effect is observed when 4SC-202 is administrated with 24h delay to chemotherapy. • Moreover, the simultaneous and sequential schedules of tested combination treatments show a synergistic pro-apoptotic and anti- clonogenistic effects, suggesting a new therapeutic avenue to increase efficacy of drugs commonly used in clinical practice. • Preliminary data suggest that 4SC-202 inhibits spheroid tumor growth consistent with decreased stem cell markers, such as CD133, NANOG and OCT4. Indeed, in many cancer types a sub-set of cancer cells, so-called cancer stem cells (CSCs) have been identified. The CSCs have been shown to be more resistant to chemotherapy and radiotherapy, providing a mechanism for disease relapse. Targeting the CSC population has highlighted a possible therapeutic window for treatment of tumors. B C Antitumoral effect of 4SC-202 as monotherapy on pancreatic cancer cell lines PANC28 Adenocarcinoma Primary Tumour KRAS P53 nt A B 4sc-202 Domatinostat the heterogeneity of PDAC patients. (B) 4SC-202 is the tosylate salt of a benzamide type HDAC inhibitor containg a N- sulfonylpyrrol scaffold with a MW of 447.5 g/mol and a pKa of 3.3. 4SC-202 shows selective inhibition of recombinant class I HDAC isoenzymes with IC50 values of about 1 μM 0.0625 0.12 5 0 . 2 5 0.5 1 2 4 8 Growth (%) 4sc-202 Domatinostat strong synergism synergism Combination Index (CI) and Dose Reduction Index (DRI) CIs < 0,9 indicated synergism DRI values represent the order of magnitude (fold) of dose reduction strong synergism synergism Combination Index (CI) and Dose Reduction Index (DRI) CIs < 0,9 indicated synergism DRI values represent the order of magnitude (fold) of dose reduction F C T R 4SC-2 0 2 C A PIRI 4SC - 202+C A PIRI 4SC - 202 - > 4 S C - 202 - > C A PIR I CTR 4SC -2 02 C APIR I 4SC - 202+C APIR I 4SC -20 2 - > 4SC - 202 -> C APIRI CTR 4SC-202 CAPI R I 4S C - 2 02+CAP IR I 4SC - 2 0 2 -> 4 SC - 20 2 - > C A P IR I CTR 4SC-2 0 2 GP 4SC-202+GP 4SC-202 -> 4 SC-202-> GP Annexin-V FOLD CHANGE C TR 4 SC - 2 02 GP 4 SC - 20 2 + GP 4SC- 20 2 -> 4 S C-202 - > GP Annexin-V FOLD CHANGE CT R 4SC-202 GP 4 SC- 20 2+ GP 4SC- 2 02 - > 4SC -2 02- > GP Annexin-V FOLD CHANGE CTR 4SC-202 CA P I RI 4SC-202+CAPIRI Absorbance 595 nm CTR 4SC-202 CA P I RI 4SC-2 0 2+CAPIRI Absorbance 595 nm CT R 4 SC-2 02 CAPI RI 4 S C-2 0 2+CAPIRI Absorbance 595 nm CT R 4 SC-2 02 GP 4 SC-20 2 + G P Absorbance 595 nm CT R 4 SC-2 02 G P 4 SC-20 2 + G P Absorbance 595 nm CTR 4SC-2 0 2 G P 4SC-202+ G P Absorbance 595 nm G Viability Assay 72h – 96h Cytotoxicity assay 48h Clonogenic assay 15 days Viability Assay 72h – 96h Cytotoxicity assay 48h Clonogenic assay 10 days B C Limiting Dilution assay 15 days D Limiting Dilution Assay 15 days H CTR 4 SC- 2 02 GP 4SC-2 02+GP CT R 4 SC- 2 0 2 G P 4SC-2 0 2+ G P CT R 4 SC- 2 02 G P 4SC-2 0 2+ G P CT R 4 SC-2 02 CAP I RI 4SC-202+CAPIRI CT R 4 SC-2 02 CAP I RI 4SC-202+CAPIRI CT R 4SC-202 CA PIR I 4 SC-2 02 + CAP I RI showed synergism in PANC28 cell line, upon simultaneous exposure to equitoxic doses (50:50 cytotoxic ratio), as shown by average values of combination indexes (CIs), lower than 0.9, calculated at 50% (CI50), of cell lethality. The combination showed a synergism in all three cell lines (strong in PANC28 cell line lower than 0.6, highlighted in red), upon a sequential treatment approach. In details, 4SC-202 was administrated with 24h delay to low dose of chemotherapy. In the lower table, as above, we evaluated the combination of 4SC-202 plus gemcitabine/taxolo. The sequential approach showed synergism in PANC1 cell line, but not in PANC28 and AspC1 cell lines. Although the anti- proliferative effect was not clear in PANC28 and AspC1, we demonstrated a clear increase of the apoptotic effect, measured as AnnexinV exposition, induced by the combination and sequential setting, compared with control and the single agents in all three PDAC cancer cell lines. The drug dose are, respectively, 4SC-202 0.5 µM, gemcitabine 50nM and paclitaxel 5nM (B). (C-D) Similarly, we demonstrated a strong synergistic effect of 4SC-202 plus gemcitabine/paclitaxel in long term treatment assay, such as clonogenistic and limiting dilution assays. (C) The drug dose are for clonogenistic assay, respectively, 4SC-202 0.12 µM, gemcitabine 2.5nM and paclitaxel 0.25nM. (D) The drug dose are for limiting dilution assay, respectively, 4SC-202 0.5 µM, gemcitabine 25nM and paclitaxel 2.5nM. Importantly, both assays evaluate the combination setting to target the clonogenistic capability of cancer cells, but in a different treatment setting. Indeed, the single cells were constantly under treatment in (C) while the cells were treated for 24h and the seeded as single cells in (D). synergism Maria Serena Roca: s.roca @istitutotumori.na.it Alfredo Budillon: a.budillon@istitutotumori.na.it METHODS Correspondence to: Figure 2. 4SC-202 affects tumorigenesis of adenocarcinoma pancreatic cells. A-B) 4SC-202 , administered as single agent, was able to inhibit tumor growth, evaluated by both antiproliferative effects (A) and ability to prevent clonogenic formation (B) from single cells. Latter assay is used to indicate a greater aggressiveness of cancer cells. In details, upon 96h of treatment, as assessed by SRB colorimetric assay, 4SC-202 inhibited growth at micromolar concentrations as reported in table inserted in panel B. Concomitantly, after 10 days, low dose of 4SC-202 (0.6µM) dramatically reduced the number of clones in all three cell lines studied.C-D) With the aim to deeply investigate the mechanism underlying the observed anti-proliferative effects of 4SC-202, we studied the impact of the drug on cell cycle and apoptosis as well. By citofluorimetric assay, we observed that 4SC-202, after only 24h of treatment, altered cell cycle, inducing an increase of G2 phase, as previously reported for colonrectal cancer cells (Zhijun H et al, Tumour Biol, 2016). Furthermore, in PANC1 and in PANC28, over the increasing doses of drug, a clear switch from G1 to G2 phases was evident (C). The perturbation of cell cycle seems also associated to an increase of apoptosis. Indeed, in all three cell lines, a clear increase of apoptosis, mesaured by FACS analysis as increase of Annexin V-FITC, was observed after 48h of treatment with 2µM of 4SC-202. Interestingly, particularly in the PANC1, 4SC-202 induced a clear increase of necrosis, suggesting that it could affect the so called phenomenon of necroptosis (D). Cell Culture The PDAC cancer cell lines are cultured in DMEM medium 10% FBS (Gibco) Cell proliferation assay Cell proliferation assay was measured in 96-well plates by a spectrophotometric dye incorporation assay (sulforhodamine B). Drugs combination studies. Drugs combination studies were based on concentration-effect curves generated as a plot of the fraction of unaffected (surviving) cells versus drug concentration (Chou T.C. and Talalay P., Adv. Enzyme Regul. 22: 27, 1984) after 72h of treatment. Combination index (CI) values of < 1, 1, and > 1 indicate synergy, additivity, and antagonism, respectively (Chou T.C. et al. J Natl Cancer Inst 1994; 86: 1517-1524). Assessment of synergy was performed quantitating drug interaction by CalcuSyn computer program (Biosoft). Cell cycle kinetic and Apoptosis. Nuclear DNA staining was performed by propidium iodide (PI). DNA-flow cytometry was performed by a FACScan flow cytometer (Becton Dickinson) acquiring 20000 events for each sample. The percentage of apoptotic cells was calculated in the sub-diploid region of the DNA content, registered as FL2 signals in linear scale. Clonogenic assay. Single cell suspensions were plated at 250-300 cells/well in 6 well/plates. After 10-15 days colonies were visualized with crystal violet in 20% methanol . Limiting dilution assay The self‐renewal capacity of colon CSCs was assayed by dissociation of colon cancer spheroids. Colon CSCs were cultured in adhesion with indicated concentrations VPA after which cells were collected and plated at serial dilution (1, 2, 4, 8, 16, 32, 64 and 128 cells/ well) in 96 well microplates with flat bottom and repellent surface for low attachment using a FACS Aria. RNA expression was evaluated by Real-time quantitative PCR using TaqMan assays or SYBR-Green-I method. 3D microtissues assay was performed by the GravityPLUS™ Hanging Drop System (InSphero) with the established prostate cell lines 22RV-1 and DU145-R80. Cells were plated with or without drugs following instructions from InSphero kit. 3D microtissues were mantained for 96h in a 37°C, 5% CO2 incubator and after which were scored with CellTiter- Glo® 3D Cell Viability Assay (Promega) by measuring the luminescence in a Multilabel Reader VICTOR X4 2030 (PerkinElmer). Fluorescence intensity DNA conten D 4 4 4 4SC-202 effects on cancer stem cell-enriched population PANC28 PANC1 AspC1 - 4SC-202 0.5µM pixel/µm 2 pixel/µm 2 A The same approach has been used to evaluate the 4SC-202 plus combination CAPIRI. In (E), the combination 4SC-202/5DFUR and 4SC-202/CAPIRI showed a strong synergism when 4SC-202 was administrated with 24h delay to chemotherapy in PANC1, PANC28 and AspC1 cell lines, suggesting that the HDAC inhibition sensitizes a specific cancer cell population before resistance to chemotherapy. In line, we demonstrated a clear cytotoxic effect in the triple combination in both simultaneous and sequential setting of treatment compared with control and the single agents in all cancer cell lines. The drug dose are, respectively, 4SC-202 0.5 µM, 5’DFUR 10µM and irinotecan 10nM (F). As for GP treatment, the 4SC-202/CAPIRI combination reduced the clonogenic capability of cancer cell line, evaluated in a clonogenic and a limiting dilution assays(G- H). (G) The drug dose are for clonogenic assay, respectively, 4SC-202 0.12 µM, 5’DFUR 0.25µM and irinotecan 0.25nM . (D) The drug dose are for limiting dilution assay, respectively, 4SC-202 0.5 µM, 5’DFUR 10µM and irinotecan 10nM. CD133 + cells Fold Change Figure 3. 4SC-202 affects the innate stem cell properties. Based on previous observations about targeting of specific population, we tested 4SC-202 in targeting of innate stem cell properties. (A) We showed that 4SC-202 inhibits pancreatic cancer 3D-self-assembled spheroids growth, underlying the ability to target the cancer stem cells (CSC) sub-population that allows spheroids formation. In details, the cells are seed and after three days they are treated with 4SC-202 (0.5µM) for 5 days and then photos are taken to evaluate the size. (B) This result is consistently associated with a decreased in expression levels of the membrane protein CD133, CSC marker upon a treatment of 4SC-202 1µM and 2µM for 24h, evaluated by cytofluorimetry. The effect is in PANC1 and AspC1 more clear then in PANC28 (C) Consistently, a significant drop in mRNA expression levels of stem cell marker NANOG and OCT4 were observed upon increasing doses of 4SC-202 (1µM-2µM) for 24h in PANC1 and AspC1 cell lines. Conversely, an increase of OCT4 and NANOG was found in PANC28 cell lines, suggesting an alternative molecular mechanism to sensitize CSC population. B NANOG OCT4 C