Joanne M. Munck

Chemosensitization of Cancer Cells by KU-0060648, A Dual Inhibitor of DNA-PK and PI-3K

DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by topoisomerase II poisons. Nonhomologous end joining (NHEJ) is a major pathway for DSB repair and requires DNA-dependent protein kinase (DNA-PK) activity. DNA-PK catalytic subunit (DNA-PKcs) is structurally similar to PI-3K, which promotes cell survival and proliferation and is upregulated in many cancers. KU-0060648 is a dual inhibitor of DNA-PK and PI-3K in vitro. KU-0060648 was investigated in a panel of human breast and colon cancer cells. The compound inhibited cellular DNA-PK autophosphorylation with IC50 values of 0.019 μmol/L (MCF7 cells) and 0.17 μmol/L (SW620 cells), and PI-3K–mediated AKT phosphorylation with IC50 values of 0.039 μmol/L (MCF7 cells) and more than 10 μmol/L (SW620 cells). Five-day exposure to 1 μmol/L KU-0060648 inhibited cell proliferation by more than 95% in MCF7 cells but only by 55% in SW620 cells. In clonogenic survival assays, KU-0060648 increased the cytotoxicity of etoposide and doxorubicin across the panel of DNA-PKcs–proficient cells, but not in DNA-PKcs–deficient cells, thus confirming that enhanced cytotoxicity was due to DNA-PK inhibition. In mice bearing SW620 and MCF7 xenografts, concentrations of KU-0060648 that were sufficient for in vitro growth inhibition and chemosensitization were maintained within the tumor for at least 4 hours at nontoxic doses. KU-0060648 alone delayed the growth of MCF7 xenografts and increased etoposide-induced tumor growth delay in both in SW620 and MCF7 xenografts by up to 4.5-fold, without exacerbating etoposide toxicity to unacceptable levels. The proof-of-principle in vitro and in vivo chemosensitization with KU-0060648 justifies further evaluation of dual DNA-PK and PI-3K inhibitors. Mol Cancer Ther; 11(8); 1789–98. ©2012 AACR.

Inhibition of HSP90 by AT13387 Delays the Emergence of Resistance to BRAF Inhibitors and Overcomes Resistance to dual BRAF and MEK Inhibition in Melanoma Models

Emergence of clinical resistance to BRAF inhibitors, alone or in combination with MEK inhibitors, limits clinical responses in melanoma. Inhibiting HSP90 offers an approach to simultaneously interfere with multiple resistance mechanisms. Using the HSP90 inhibitor AT13387, which is currently in clinical trials, we investigated the potential of HSP90 inhibition to overcome or delay the emergence of resistance to these kinase inhibitors in melanoma models. In vitro, treating vemurafenib-sensitive cells (A375 or SK-MEL-28) with a combination of AT13387 and vemurafenib prevented colony growth under conditions in which vemurafenib treatment alone generated resistant colonies. In vivo, when AT13387 was combined with vemurafenib in a SK-MEL-28, vemurafenib-sensitive model, no regrowth of tumors was observed over 5 months, although 2 of 7 tumors in the vemurafenib monotherapy group relapsed in this time. Together, these data suggest that the combination of these agents can delay the emergence of resistance. Cell lines with acquired vemurafenib resistance, derived from these models (A375R and SK-MEL-28R) were also sensitive to HSP90 inhibitor treatment; key clients were depleted, apoptosis was induced, and growth in 3D culture was inhibited. Similar effects were observed in cell lines with acquired resistance to both BRAF and MEK inhibitors (SK-MEL-28RR, WM164RR, and 1205LuRR). These data suggest that treatment with an HSP90 inhibitor, such as AT13387, is a potential approach for combating resistance to BRAF and MEK inhibition in melanoma. Moreover, frontline combination of these agents with an HSP90 inhibitor could delay the emergence of resistance, providing a strong rationale for clinical investigation of such combinations in BRAF-mutated melanoma. Mol Cancer Ther; 13(12); 2793–804. ©2014 AACR.

The Novel, Small-Molecule DNA Methylation Inhibitor SGI-110 as an Ovarian Cancer Chemosensitizer

Purpose: To investigate SGI-110 as a “chemosensitizer” in ovarian cancer and to assess its effects on tumor suppressor genes (TSG) and chemoresponsiveness-associated genes silenced by DNA methylation in ovarian cancer. Experimental Design: Several ovarian cancer cell lines were used for in vitro and in vivo platinum resensitization studies. Changes in DNA methylation and expression levels of TSG and other cancer-related genes in response to SGI-110 were measured by pyrosequencing and RT-PCR. Results: We demonstrate in vitro that SGI-110 resensitized a range of platinum-resistant ovarian cancer cells to cisplatin (CDDP) and induced significant demethylation and reexpression of TSG, differentiation-associated genes, and putative drivers of ovarian cancer cisplatin resistance. In vivo, SGI-110 alone or in combination with CDDP was well tolerated and induced antitumor effects in ovarian cancer xenografts. Pyrosequencing analyses confirmed that SGI-110 caused both global (LINE1) and gene-specific hypomethylation in vivo, including TSGs (RASSF1A), proposed drivers of ovarian cancer cisplatin resistance (MLH1 and ZIC1), differentiation-associated genes (HOXA10 and HOXA11), and transcription factors (STAT5B). Furthermore, DNA damage induced by CDDP in ovarian cancer cells was increased by SGI-110, as measured by inductively coupled plasma-mass spectrometry analysis of DNA adduct formation and repair of cisplatin-induced DNA damage. Conclusions: These results strongly support further investigation of hypomethylating strategies in platinum-resistant ovarian cancer. Specifically, SGI-110 in combination with conventional and/or targeted therapeutics warrants further development in this setting. Clin Cancer Res; 20(24); 6504–16. ©2014 AACR.

DNA-PK – a candidate driver of hepatocarcinogenesis and tissue biomarker that predicts response to treatment and survival

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.

Abstract 2944: AT-IAP, a dual cIAP1 and XIAP antagonist with oral antitumor activity in melanoma models.

Melanoma is a highly aggressive malignancy with an exceptional ability to develop resistance and no curative therapy is available for patients with metastatic disease. Inhibitor of apoptosis proteins (IAP) play a key role in preventing cell death by apoptosis. In normal cell, IAPs are highly regulated by endogenous antagonists (e.g. SMAC) but in melanoma cell lines and in patient samples expression levels of IAPs are generally high and depleting IAPs by siRNA tended to reduce cell viability, with XIAP reduction being the most efficient [1]. Small molecule IAP antagonists have the ability to switch IAP-controlled pro-survival pathways towards apoptosis and cell death. Recent evidence suggests that a true dual antagonist of both cIAP1 and XIAP will promote an effective apoptotic response through generation of death-inducing ripoptosome complexes, with resultant caspase activation [2, 3]. We have used our fragment-based drug discovery technology PyramidTM to derive a non-peptidomimetic IAP antagonist, AT-IAP, which does not have an alanine warhead and has nanomolar cellular potency for both XIAP and cIAP1. Initial pharmacokinetic and pharmacodynamic modeling of AT-IAP in mice bearing the MDA-MB-231 cell line indicated that daily oral dosing of AT-IAP at 30 mg/kg ensures high concentrations of compound in tumor and plasma over a 24 h period with resultant inhibition of both XIAP and cIAP1 and induction of apoptosis markers (cleaved PARP and cleaved caspase-3). In this paper, we describe the characterization of AT-IAP in melanoma models. An in vitro cell line proliferation screen demonstrated that 36% of melanoma cell lines exhibited enhanced sensitivity to AT-IAP, which was improved on addition of exogenous 1 ng/ml TNF-α (92% of cell lines were sensitive to AT-IAP + TNF-α). Sensitivity of melanoma cells to AT-IAP has also been confirmed in a panel of 20 primary melanoma tumors in colony formation assays set up in the presence and absence of added TNF-α. Finally, a set of biomarkers has been identified and used to predict single agent activity of AT-IAP in a range of melanoma cell line and patient derived xenograft models. [1] Engesaeter et al., Cancer Biology & Therapy, 2011, 12 (1), 47 [2] Ndubaku et al., ACS Chem Biol., 2009, 4 (7), 557 [3] Meier, P., Nat Rev. Cancer, 2010, 10 (8), 561 Citation Format: Gianni Chessari, Ahn Maria, Ildiko Buck, Elisabetta Chiarparin, Joe Coyle, James Day, Martyn Frederickson, Charlotte Griffiths-Jones, Keisha Hearn, Steven Howard, Tom Heightman, Petra Hillmann, Aman Iqbal, Christopher N. Johnson, Jon Lewis, Vanessa Martins, Joanne Munck, Mike Reader, Lee Page, Anna Hopkins, Alessia Millemaggi, Caroline Richardson, Gordon Saxty, Tomoko Smyth, Emiliano Tamanini, Neil Thompson, George Ward, Glyn Williams, Pamela Williams, Nicola Wilsher, Alison Woolford. AT-IAP, a dual cIAP1 and XIAP antagonist with oral antitumor activity in melanoma models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2944. doi:10.1158/1538-7445.AM2013-2944

ASTX660, a Novel Non-peptidomimetic Antagonist of cIAP1/2 and XIAP, Potently Induces TNFα-Dependent Apoptosis in Cancer Cell Lines and Inhibits Tumor Growth

Because of their roles in the evasion of apoptosis, inhibitor of apoptosis proteins (IAP) are considered attractive targets for anticancer therapy. Antagonists of these proteins have the potential to switch prosurvival signaling pathways in cancer cells toward cell death. Various SMAC-peptidomimetics with inherent cIAP selectivity have been tested clinically and demonstrated minimal single-agent efficacy. ASTX660 is a potent, non-peptidomimetic antagonist of cIAP1/2 and XIAP, discovered using fragment-based drug design. The antagonism of XIAP and cIAP1 by ASTX660 was demonstrated on purified proteins, cells, and in vivo in xenograft models. The compound binds to the isolated BIR3 domains of both XIAP and cIAP1 with nanomolar potencies. In cells and xenograft tissue, direct antagonism of XIAP was demonstrated by measuring its displacement from caspase-9 or SMAC. Compound-induced proteasomal degradation of cIAP1 and 2, resulting in downstream effects of NIK stabilization and activation of noncanonical NF-κB signaling, demonstrated cIAP1/2 antagonism. Treatment with ASTX660 led to TNFα-dependent induction of apoptosis in various cancer cell lines in vitro, whereas dosing in mice bearing breast and melanoma tumor xenografts inhibited tumor growth. ASTX660 is currently being tested in a phase I–II clinical trial (NCT02503423), and we propose that its antagonism of cIAP1/2 and XIAP may offer improved efficacy over first-generation antagonists that are more cIAP1/2 selective. Mol Cancer Ther; 17(7); 1381–91. ©2018 AACR.

Fragment-Based Discovery of a Potent, Orally Bioavailable Inhibitor That Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

Aberrant activation of the MAPK pathway drives cell proliferation in multiple cancers. Inhibitors of BRAF and MEK kinases are approved for the treatment of BRAF mutant melanoma, but resistance frequently emerges, often mediated by increased signaling through ERK1/2. Here, we describe the fragment-based generation of ERK1/2 inhibitors that block catalytic phosphorylation of downstream substrates such as RSK but also modulate phosphorylation of ERK1/2 by MEK without directly inhibiting MEK. X-ray crystallographic and biophysical fragment screening followed by structure-guided optimization and growth from the hinge into a pocket proximal to the C-α helix afforded highly potent ERK1/2 inhibitors with excellent kinome selectivity. In BRAF mutant cells, the lead compound suppresses pRSK and pERK levels and inhibits proliferation at low nanomolar concentrations. The lead exhibits tumor regression upon oral dosing in BRAF mutant xenograft models, providing a promising basis for further optimization toward clinical pERK1/2 modulating ERK1/2 inhibitors.

Abstract 1247: ERK1/2 inhibitors with distinct modes of inhibition confer different response profiles in KRAS mutant cancers

The clinical benefit of targeting the MAPK pathway is demonstrated by the success of RAF and MEK inhibitors in the treatment of melanoma. However, response to such agents is short-lived due to the onset of resistance mechanisms that result in reactivation of ERK signalling. Additionally, many BRAF- and KRAS-mutant cancers exhibit de novo resistance. As the direct targeting of ERK may overcome the limitations of RAF or MEK inhibitors, there are several ERK1/2 inhibitors in development. SCH772984 (SCH, Merck-Schering) is an example of an ERK1/2 inhibitor that in addition to inhibiting ERK catalytic activity also inhibits the phosphorylation of residues within the ERK activation loop. Several ERK1/2 inhibitors have been described that inhibit ERK catalytic activity without modulating ERK phosphorylation e.g GDC-0994 (GDC, Genentech). Here we aim to characterise the biological relevance of the different modes of ERK inhibition. We investigated the biochemical mechanism by which SCH inhibits ERK phosphorylation and demonstrated that it prevents the phosphorylation of ERK by MEK (T202/Y204), but does not affect MEK activity. We characterised the effects of the distinct modes of ERK inhibition on ERK substrates, cellular pERK levels, pathway feedback activation and sensitivities of cell lines with different genetic backgrounds. In BRAF V600E -mutant cells e.g A375 (melanoma) and Colo205 (colorectal), 16 hr treatment with SCH and GDC significantly inhibited the phosphorylation of RSK (ERK substrate). SCH significantly inhibited ERK phosphorylation, whereas GDC did not modulate pERK levels. Neither compound induced pathway feedback activation. In proliferation assays A375 and Colo205 cells had remarkably similar sensitivities to SCH (EC 50 40 nM and 30 nM, respectively) and GDC (EC 50 = 140 nM in both). In contrast to BRAF-mutant cells, SCH and GDC treatment conferred pMEK induction in the KRAS-mutant cell line HCT116 (while still significantly inhibiting RSK phosphorylation). This is likely to be attributable to the feedback-driven C-RAF activation commonly associated with MAPK pathway inhibition in KRAS-mutant cells. In addition to pMEK, GDC also conferred an induction of pERK. However, SCH overcame pathway feedback and inhibited the ERK phosphorylation. The inhibition of pathway feedback conferred by SCH was associated with increased sensitivity of HCT116 cells to SCH (EC 50 = 30 nM) compared to GDC (EC 50 = 1.1 μM). In a cell panel screen comprising 25 BRAF-mutant and 63 KRAS-mutant cell lines, SCH and GDC had similar activity in BRAF-mutant cell lines. However, although KRAS-mutant cell lines were sensitive to SCH, they were largely resistant to GDC. These data suggest that due to their ability to overcome MAPK inhibitor-induced pathway feedback, ERK inhibitors that also inhibit the phosphorylation of ERK would be more effective in KRAS-mutant cancer than those that inhibit ERK activity alone. Citation Format: Joanne M. Munck, Brent Graham, Juan Castro, Aurelie Courtin, Sandra Muench, Sharna Rich, Harpreet K. Saini, Jessica Brothwood, John Lyons, Neil T. Thompson, Nicola G. Wallis, Chris Murray. ERK1/2 inhibitors with distinct modes of inhibition confer different response profiles in KRAS mutant cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1247.

Abstract 3756: The DNA hypomethylating agent SGI-110, reverses the platinum resistance of ovarian cancer models

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA SGI-110, is a novel second generation DNA hypomethylating agent, which shows promising single agent activity in leukaemia patients ([NCT01261312][1]) and is currently under investigation as a priming agent in solid tumors. There is evidence that the acquisition of platinum resistance in ovarian cancer is associated with DNA methylation changes that result in epigenetic silencing of specific genes. Pre-clinical studies suggest that DNA hypomethylating agents can reverse such resistance. SGI-110 is currently in a Phase II clinical trial in combination with carboplatin, in platinum-resistant recurrent ovarian cancer patients ([NCT01696032][2]). SGI-110 is a dinucleotide of decitabine and deoxyguanosine, which is resistant to modification by cytidine deaminase: a common pathway of decitabine metabolism and deactivation. We demonstrated that SGI-110 conferred global LINE1 de-methylation and re-expression of epigenetically silenced genes in a panel of ovarian cancer cell lines. We identified three cell lines, A2780, OVCAR8 and OAW28, which were sensitised to cisplatin following SGI-110 treatment (4-fold, 9-fold and 3-fold sensitisation, respectively). It has previously been demonstrated that epigenetic silencing of the mismatch repair protein MLH1 contributes to the cisplatin resistance of A2780 cells. Using pyrosequencing, we confirmed MLH1 promoter hypermethylation (96.2% methylation) and silencing of MLH1 expression in A2780 cells. Treatment with 0.1 µM SGI-110 conferred 25% MLH1 promoter de-methylation and MLH1 re-expression. We demonstrated that MLH1 is not epigenetically silenced in OVCAR8 or OAW28 cells. This suggests that the cisplatin resistance of these cell lines are due to distinct epigenetic mechanisms, which we are currently investigating. We assessed SGI-110 activity in an in vivo A2780 xenograft model. Mice bearing subcutaneous xenografts were treated with SGI-110 (2 mg/kg, s.c, daily x5), either as single agent or prior to treatment with cisplatin (2 mg/kg, or 4 mg/kg cisplatin i.p). Cisplatin treatment alone had no anti-tumor activity. Single agent SGI-110 conferred moderate anti-tumor activity. However, the combination of SGI-110 with 4 mg/kg cisplatin, conferred even greater anti-tumor activity than either agent alone (p<0.05). Since single agent cisplatin had no anti-tumor activity, this demonstrates that priming with SGI-110 reversed the intrinsic cisplatin resistance of the A2780 tumors. SGI-110 conferred 11% LINE1 de-methylation, 2.2% MLH1 promoter de-methylation and a 3-fold increase in MLH1 mRNA levels in tumors. These data suggest that SGI-110 can reverse the cisplatin resistance of ovarian cancer models by restoring the expression of epigenetically silenced genes and supports our ongoing Phase II trial evaluating SGI-110 in combination with carboplatin, in platinum-resistant recurrent ovarian cancer patients Citation Format: Joanne M. Munck, John Lyons, Neil T. Thompson, Nicola G. Wallis, Pietro Taverna. The DNA hypomethylating agent SGI-110, reverses the platinum resistance of ovarian cancer models. [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 3756. doi:10.1158/1538-7445.AM2014-3756 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01261312&atom=%2Fcanres%2F74%2F19_Supplement%2F3756.atom [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01696032&atom=%2Fcanres%2F74%2F19_Supplement%2F3756.atom

PTU-042 DNA-PK or ATM inhibition inhibits non-homologous end joining and enhances chemo- and radio sensitivity in hepatocellular cancer cell lines

Introduction Hepatocellular carcinoma (HCC) is chemotherapy resistance possibly due to dysregulation of DNA damage signalling and repair. 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). DSBs are repaired by non-homologous end joining (NHEJ), initiated by DNA-dependent protein kinase (DNA-PK), and homologous recombination (HR), reportedly initiated by Ataxia telangiectasia mutated (ATM). DNA-PK is up-regulated in HCC (GEO profiles), possibly contributing to anticancer therapy resistance. To assess DNA-PK and ATM as therapeutic targets for chemo- and radio-sensitisation in HCC we determined the effect of their inhibition in HCC cell lines. Methods DNA-PK and ATM 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). Studies were performed in the presence and absence of the DNA-PK inhibitor NU7441, and the ATM inhibitor KU55933. Results DNA-PK protein concentration and activity were high in all HCC cell lines. In contrast, ATM expression varied, and was lowest in Hep3B cells. Cell-specific sensitivities to IR and doxorubicin correlated with ATM expression (highest in HepG2 and lowest in Hep3B). NU7441 sensitised all cells to doxorubicin (average PF50 4.3±3.0) and IR (average PF50 3.9±1.1), significantly increasing growth inhibition and reducing survival (4.8 to 3.3-fold; colony forming assays). KU55933 significantly potentiated cytotoxicity in HepG2 cells (eightfold) but had little effect on cytotoxicity in Hep3B cells. Following exposure to IR, both NU7441 and KU55933 delayed DSB repair (∼50% clearance yH2AX foci at 4 h vs only 10%–15% in presence NU7441). NU7441 also enhanced HR (threefold increase in RAD51 foci), while KU55933 had little effect. Conclusion DNA-PK levels were high in all HCC cells and its inhibition with NU7441 was associated with significant chemo- and radio-sensitisation. Potentiation by ATM inhibition varied in the cell lines, reflecting the level of ATM expression. Both inhibitors substantially impaired the rapid phase of DNA repair commonly attributed to NHEJ. Notably, KU55933 had little effect on HR, suggesting that ATM is not central to this repair pathway. We propose that these inhibitors will increase the effectiveness of lower safer doses of cytotoxic therapies, amplifying tumour toxicity, and that DNA-PK and ATM levels in tumour and non-tumour liver will predict those patient likely to benefit. Competing interests None declared.