Data Supplement from DNA-PK—A Candidate Driver of Hepatocarcinogenesis and Tissue Biomarker That Predicts Response to Treatment and Survival

CDK4/6 inhibition is now part of the standard armamentarium for patients with estrogen receptor-positive (ER+) breast cancer, so that defining mechanisms of resistance is a pressing issue. Here, we identify increased CDK6 expression as a key determinant of acquired resistance after palbociclib treatment in ER+ breast cancer cells. CDK6 expression is critical for cellular survival during palbociclib exposure. The increased CDK6 expression observed in resistant cells is dependent on TGF-β pathway suppression via miR-432-5p expression. Exosomal miR-432-5p expression mediates the transfer of the resistance phenotype between neighboring cell populations. Levels of miR-432-5p are higher in primary breast cancers demonstrating CDK4/6 resistance compared to those that are sensitive. These data are further confirmed in pre-treatment and post-progression biopsies from a parotid cancer patient who had responded to ribociclib, demonstrating the clinical relevance of this mechanism. Finally, the CDK4/6 inhibitor resistance phenotype is reversible in vitro and in vivo by a prolonged drug holiday.

CDK4 is emerging as a target in KRAS-mutant non-small cell lung cancer (NSCLC). We demonstrate that KRAS-mutant NSCLC cell lines are initially sensitive to the CDK4/6 inhibitor palbociclib, but readily acquire resistance associated with increased expression of CDK6, D-type cyclins and cyclin E. Resistant cells also demonstrated increased ERK1/2 activity and sensitivity to MEK and ERK inhibitors. Moreover, MEK inhibition reduced the expression and activity of cell cycle proteins mediating palbociclib resistance. In resistant cells, ERK activated mTOR, driven in part by upstream FGFR1 signaling resulting from the extracellular secretion of FGF ligands. A genetically-engineered mouse model of KRAS-mutant NSCLC initially sensitive to palbociclib similarly developed acquired resistance with increased expression of cell cycle mediators, ERK1/2 and FGFR1. In this model, resistance was delayed with combined palbociclib and MEK inhibitor treatment. These findings implicate an FGFR1-MAP kinase-mTOR pathway resulting in increased expression of D-cyclins and CDK6 that confers palbociclib resistance and indicate that CDK4/6 inhibition acts to promote MAP kinase dependence.

Abstract Multiple potent and highly selective inhibitors of the cell cycle kinases - CDK4 and CDK6 are in development. One such inhibitor, palbociclib, was recently approved for use in combination with letrozole for the treatment of estrogen receptor positive (ER+) breast cancer. Cyclin D-dependent kinase activity is a driving factor for ER+ breast carcinogenesis, irrespective of CCND1 amplification, making CDK4/6 inhibition a promising approach for this breast cancer subset. However, as with all cancer treatments, resistance will be a major issue limiting the efficacy of this approach. To date, mechanisms of palbociclib resistance have not been extensively investigated. Through interrogation of the generated CDK4/6 inhibition resistant cells we discovered overexpression of CDK6, specifically, mediates resistance. CDK6 depletion reversed resistance and overexpression caused resistance, whereas the same was not true when CDK4 or D cyclin protein levels were manipulated. Interestingly, when generating and analyzing resistant cell populations, we observed a “bystander effect”, by which resistance was transmissible between cells. The “resistant bystander” cells display all characteristics of the drug exposure induced resistant cells, however became resistant in just 48 hours as oppose to 14 weeks. Analysis of conditioned medium revealed that the transmission of resistance is dependent on exosomes, but not protein or DNA. We identified a specific miRNA, present in the exosomes of resistant cells, by microarray which caused resistance when excreted. Additionally, overexpression and inhibition of the miRNA confirmed that it is responsible for causing resistance. miRNA overexpressing cells exhibited the same phenotype as drug induced resistant cells, and furthermore, could cause resistance in neighboring cell populations by exosome mediated signaling. Using Biotin labelled miRNA-mRNA pulldown followed by RNA-seq, we identified the TGFβ pathway as the miRNA target. Downregulation of the TGFβ caused a decrease in the CDK inhibitors, p15 and p21, resulting in an increased CDK6 protein level and palbociclib resistance. We subsequently confirmed these data in patient samples by comparing before treatment and post relapse biopsies. These findings highlight a novel mechanism of conferred drug resistance as well as new insights into acquired CDK4/6 inhibitor resistance. Citation Format: Liam Cornell, Geoffrey I. Shapiro. MicroRNA mediated CDK4/6 inhibitor resistance via extracellular signaling [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 2347. doi:10.1158/1538-7445.AM2017-2347

The eukaryotic cell cycle is a tightly regulated series of events coordinated by the periodic activation of members of the cyclin dependent kinases (CDK) family. In addition, a subset of CDK family members play critical roles in transcriptional regulation. Dysregulation of CDK activity by a variety of genetic and epigenetic mechanisms is universally observed in cancer and is thought to be a primary driving force in carcinogenesis, such that there has been longstanding interest in targeting CDKs for cancer therapy.Along with orchestrating the cell cycle and transcriptional events, CDKs have also been directly implicated in the DNA damage response. CDK activity governs cell cycle phase and thus indirectly affects double strand break (DSB) repair pathway choice. Recent evidence also directly implicates CDKs 1 and 2 in homologous recombination DNA repair (HRR) since their activities are crucial at early stages of the repair pathway. These findings suggest that CDK inhibition may not only address aberrant cell proliferation, but may also sensitize cells to a variety of DNA damaging agents as well as PARP inhibition.

Abstract Purpose: Therapy resistance and associated liver disease make hepatocellular carcinomas (HCC) difficult to treat with traditional cytotoxic therapies, whereas newer targeted approaches offer only modest survival benefit. We focused on DNA-dependent protein kinase, DNA-PKcs, encoded by PRKDC and central to DNA damage repair by nonhomologous end joining. Our aim was to explore its roles in hepatocarcinogenesis and as a novel therapeutic candidate. Experimental Design: PRKDC was characterized in liver tissues from of 132 patients [normal liver (n = 10), cirrhotic liver (n = 13), dysplastic nodules (n = 18), HCC (n = 91)] using Affymetrix U133 Plus 2.0 and 500 K Human Mapping SNP arrays (cohort 1). In addition, we studied a case series of 45 patients with HCC undergoing diagnostic biopsy (cohort 2). Histological grading, response to treatment, and survival were correlated with DNA-PKcs quantified immunohistochemically. Parallel in vitro studies determined the impact of DNA-PK on DNA repair and response to cytotoxic therapy. Results: Increased PRKDC expression in HCC was associated with amplification of its genetic locus in cohort 1. In cohort 2, elevated DNA-PKcs identified patients with treatment-resistant HCC, progressing at a median of 4.5 months compared with 16.9 months, whereas elevation of activated pDNA-PK independently predicted poorer survival. DNA-PKcs was high in HCC cell lines, where its inhibition with NU7441 potentiated irradiation and doxorubicin-induced cytotoxicity, whereas the combination suppressed HCC growth in vitro and in vivo. Conclusions: These data identify PRKDC/DNA-PKcs as a candidate driver of hepatocarcinogenesis, whose biopsy characterization at diagnosis may impact stratification of current therapies, and whose specific future targeting may overcome resistance. Clin Cancer Res; 21(4); 925–33. ©2014 AACR.

peptidase IV-inhibitor, on hepatic inflammation and fibrosis in models of biliary fibrosis and of NASH.Methods: Linagliptin was tested in macrophage and myofibroblast cultures, and administered daily by gavage at 0.5, 5, 10 and 50 mg daily per kg BW to Mdr2KO mice from week 7-11 of age, and to 8 week old C57BL/6 mice fed a methionine and choline deficient diet (MCD) for 8 weeks.Wildtype mice fed a supplemented diet served as controls, respectively.Hepatic collagen was measured biochemically, serum biochemistries were determined by an autoanalyzer.Fibrosis and inflammation related transcript levels were quantified from livers by real-time qRT-PCR.Liver histology was assessed by connective tissue staining and histochemistry for inflammation and alpha-smooth muscle actin (alpha-SMA).Results: In Mdr2KO mice, 10 and 50 mg/kg/day of Linagliptin increased putatively anti-fibrotic MMP-9 and -13, but decreased procollagen a1(I) and TGFb1, TIMP-1, MMP-8 transcript levels.However, in vitro macrophage or fibroblast differentiation, and their production of proinflammatory or profibrogenic transcripts were unaffected.Mice fed the MCD diet showed a rapid induction of hepatic steatosis, inflammation and a 2-fold increase in fibrosis compared to controls, with an up to 10-fold upregulation of procollagen a1(I), TGFb1, aSMA, MMP-3 and -9, TIMP-1, CCL3 and TNFa mRNA expression.Linagliptin lowered serum ALT, AST, ALP and triglycerides, but did not affect hepatic collagen accumulation in both the Mdr2KO and the MCD model. Conclusion:In experimental biliary fibrosis (Mdr2KO mice) and in a surrogate NASH model (MCD) oral Linagliptin was well tolerated and lowered parameters of (hepatocyte) inflammation.Linagliptin demonstrated only a modest direct antifibrotic effect at a higher dose, possibly due to an inhibitory effect on fibroblast activation protein (FAP) rather than via induction of GLP-1.

Abstract Background DNA double-strand breaks (DSBs), the most cytotoxic lesions induced by ionizing radiation (IR) and anticancer drugs such as topoisomerase II poisons (e.g., doxorubicin), are repaired by non-homologous end joining (NHEJ) and homologous recombination (HR). DNA-dependent protein kinase (DNA-PK), which initiates NHEJ, is up-regulated in hepatocellular carcinoma (HCC) (GEO profiles), possibly contributing to anticancer therapy resistance. To assess DNA-PK as a potential therapeutic target for chemo- and radio-sensitisation in HCC we determined the effect of the DNA-PK inhibitor, NU7441, on DSB repair and cytotoxicity in HCC cells. Methods DNA-PK protein levels and activation by IR (Western blot), DSB levels (y-H2AX foci), HR (RAD51 foci), cell growth (DAPI fluorescence) and cytotoxicity (colony formation) following exposure to IR or doxorubicin was determined in a panel of 6 hepatoma cell lines (HepG2, Hep3B, Huh7, SNU-182, SNU475 and PLC/PRF/5). Results DNA-PK protein concentration and activity did not vary significantly across the panel (±24% and ±37%, respectively) but there were cell-specific sensitivities to IR and doxorubicin (e.g. HepG2 2-fold more resistant than Hep3B). NU7441 significantly sensitised all cells to both doxorubicin (average PF50 4.3±3.0) and IR (average PF50 3.9±1.1) in growth inhibition assays and significantly reduced survival (4.8 to 3.3-fold) in colony forming assays. Following exposure to IR, NU7441 significantly delayed yH2AX focus clearance in all cell lines (e.g. only 13% cleared at 4 hr compared to 50% in control), but increased RAD51 focus formation (3-fold). Conclusion While chemo- and radio-sensitivity in HCC cells was not dependent on DNA-PK expression or activity, NU7441 causes greater than 2-fold chemo- and radiosensitisation in all cells. This was accompanied by a substantial reduction in the rapid phase of DNA repair (NHEJ-dependent) and a shift to a greater reliance on the slower HR repair. DNA-PK inhibitors may have potential as chemo- and radio-sensitisors in hepatoma patients. 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 3122. doi:1538-7445.AM2012-3122

DNA double-strand breaks (DSBs) are the most cytotoxic lesions induced by ionising radiation (IR) and anticancer drugs, such as topoisomerase II poisons (eg, doxorubicin). The major DSB repair pathways are non-homologous end joining (NHEJ) and homologous recombination (HR), in which DNA-Dependent Protein Kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) are key components. DNA-PK in particular is up-regulated in hepatocellular carcinoma, (GEO profiles) possibly contributing to resistance to cytotoxic therapies. To assess DNA-PK and ATM as therapeutic targets for chemo- and radio-sensitisation in hepatoma. Basal protein levels and activities were determined by Western blot analysis in hepatoma cell lines. DNA-PK and ATM activity following doxorubicin stimulation was measured using antibodies specific to phosphorylated Ser-2056 DNA-PKcs and phosphorylated Ser-1981 ATM. DSB repair was measured by immunofluorescence detection of γ-H2AX foci. Cell survival was determined by clonogenic assay. We demonstrated high basal levels of DNA-PK in three hepatoma cell lines (Huh7, Hep3B and HepG2), with DNA-PK activation induced by 0.25 μM doxorubicin. Despite similar DNA-PK activation, we observed differential sensitivity to doxorubicin (7%, 49% and 75% survival at 10 nM doxorubicin in Huh7, Hep3B and HepG2, respectively). HepG2 cells with the greatest resistance to doxorubicin displayed a 10-fold activation of ATM relative to the other cell lines. The DNA-PK inhibitor NU7441, increased doxorubicin and ionising radiation (IR) induced cytotoxicity in all cell lines (1.3 up to fourfold), correlating with a reduction in DSB repair measured by γ-H2AX foci. Importantly, in doxorubicin resistant HepG2 cells, while incubation with NU7441 or the ATM inhibitor (KU55933) alone, had minimal effects on cell survival (91% and 86%, respectively), their combination in the absence of a cytotoxic agent markedly inhibited cell survival (21%; p<0.001, ANOVA). The addition of 10 nM doxorubicin reduced survival to less than 5% of colonies. These findings support the clinical application of DNA-PK and ATM inhibitors as chemo- and radio-sensitisors in hepatoma patients. Furthermore, these data suggest that hepatoma cell survival is dependent on up-regulation of DSB repair, effected by either DNA-PK or ATM, and that inhibition of both induces synthetic lethality—preventing DSB repair by both NHEJ and HR. The therapeutic implication is that in combination, these agents could be used to specifically induce cancer cell death, with minimal toxicity to surrounding liver tissues.