Craig MacKay

Biochemical characterisation of the SWI/SNF family member HLTF

HLTF is highly similar in domain organisation to yeast Rad5. We identify PTIP and RPA70, both involved in DNA replication and DNA repair, as HLTF-interacting proteins although cells depleted of HLTF did not show defects in cellular responses to DNA damage. In vitro, HLTF has ATPase activity and E3 ubiquitin ligase activity with a range of E2 ubiquitin conjugating enzymes. HLTF expression is severely reduced in a range of cancer cells, and we suggest that the HLTF antibodies generated in this study could be used for cancer diagnostic purposes.

E3 ubiquitin ligase HOIP attenuates apoptotic cell death induced by cisplatin.

The genotoxin cisplatin is commonly used in chemotherapy to treat solid tumors, yet our understanding of the mechanism underlying the drug response is limited. In a focused siRNA screen, using an siRNA library targeting genes involved in ubiquitin and ubiquitin-like signaling, we identified the E3 ubiquitin ligase HOIP as a key regulator of cisplatin-induced genotoxicity. HOIP forms, with SHARPIN and HOIL-1L, the linear ubiquitin assembly complex (LUBAC). We show that cells deficient in the HOIP ligase complex exhibit hypersensitivity to cisplatin. This is due to a dramatic increase in caspase-8/caspase-3-mediated apoptosis that is strictly dependent on ATM-, but not ATR-mediated DNA damage checkpoint activation. Moreover, basal and cisplatin-induced activity of the stress response kinase JNK is enhanced in HOIP-depleted cells and, conversely, JNK inhibition can increase cellular resistance to cisplatin and reverse the apoptotic hyperactivation in HOIP-depleted cells. Furthermore, we show that HOIP depletion sensitizes cancer cells, derived from carcinomas of various origins, through an enhanced apoptotic cell death response. We also provide evidence that ovarian cancer cells classified as cisplatin-resistant can regain sensitivity following HOIP downregulation. Cumulatively, our study identifies a HOIP-regulated antiapoptotic signaling pathway, and we envisage HOIP as a potential target for the development of combinatorial chemotherapies to potentiate the efficacy of platinum-based anticancer drugs.

The Fanconi Anaemia Components UBE2T and FANCM Are Functionally Linked to Nucleotide Excision Repair

The many proteins that function in the Fanconi anaemia (FA) monoubiquitylation pathway initiate replicative DNA crosslink repair. However, it is not clear whether individual FA genes participate in DNA repair pathways other than homologous recombination and translesion bypass. Here we show that avian DT40 cell knockouts of two integral FA genes – UBE2T and FANCM are unexpectedly sensitive to UV-induced DNA damage. Comprehensive genetic dissection experiments indicate that both of these FA genes collaborate to promote nucleotide excision repair rather than translesion bypass to protect cells form UV genotoxicity. Furthermore, UBE2T deficiency impacts on the efficient removal of the UV-induced photolesion cyclobutane pyrimidine dimer. Therefore, this work reveals that the FA pathway shares two components with nucleotide excision repair, intimating not only crosstalk between the two major repair pathways, but also potentially identifying a UBE2T-mediated ubiquitin-signalling response pathway that contributes to nucleotide excision repair.

Abstract P5-03-10: CYC065, a novel CDK2/9 inhibitor: Molecular basis for clinical development in basal-like triple-negative breast cancer

CYC065 is a novel CDK inhibitor, which inhibits CDK2 and 9 with IC50 values of 5 and 26 nM, respectively. Following completion of IND-enabling studies, CYC065 has been cleared by FDA for first-in-human Phase 1 clinical trials. Triple-negative breast cancers (TNBC), particularly the basal subtype, often exhibit aggressive characteristics. Despite good initial responses to chemotherapy, patients experience early relapse and diminished 5 year survival. Molecular features of basal-like TNBC include amplification or overexpression of cyclin E and MYC, suggesting potential utility for a CDK2/9 inhibitor such as CYC065. CYC065 is effective in cyclin E-overexpressing tumors, such as uterine serous carcinoma1 and trastuzumab-resistant Her2+ breast cancer2. Moreover, CDK inhibition has also been reported to be synthetic lethal with overexpressed MYC3. This led us to assess the potency and mechanism of action of CYC065 in basal-like TNBC models to evaluate the potential for CYC065 development in this indication. In vitro cell-based experiments support twice weekly pulse dosing using submicromolar concentrations of CYC065 to achieve maximum impact on cell growth in the majority of breast cancer cell lines tested. Preclinical toxicology data indicate that such levels and durations of exposure are achievable and well tolerated. As a single agent, CYC065 treatment in breast cancer cells resulted in inhibition of RNA-Pol II phosphorylation, down-regulation of Mcl-1, up-regulation of p53 and rapid induction of apoptosis. The impact of CYC065 on CDK2 targets, cyclin E and MYC was also explored. Interestingly immortalized cell lines obtained from non-malignant tissue displayed similar effects on RNA Pol II, Mcl-1, and p53 but did not undergo apoptosis and consequently exhibited relative resistance to CYC065, indicative of a potential therapeutic window. Cell cycle analysis demonstrated that CYC065 treatment induced an increase in G1 population with no significant induction of cell death in non-malignant derived cell lines, compared to cancer cell lines, in which there was significant induction of cell death. CDKs have a role in DNA repair which can be exploited to enhance the effectiveness of DNA damaging agents. Seliciclib, an oral, first generation CDK2/9 inhibitor, can be effectively combined with DNA damaging agents, such as the oral nucleoside analogue sapacitabine, or its active metabolite CNDAC. A Phase 1 clinical trial is currently underway to evaluate this combination (NCT00999401). Similarly to seliciclib, we demonstrate that CYC065 is synergistic in combination with CNDAC when given sequentially across multiple breast cancer cell lines. CNDAC-induced double strand breaks persisted for longer when cells were subsequently treated with CYC065, supporting the conclusion that these CDK inhibitors suppress DNA double-strand break repair capacity, which may contribute to the observed synergy. Taken together the data establish CYC065 as a promising anti-cancer agent in basal-like TNBC, with the potential to be combined effectively in this indication with DNA damaging agents. 1. Cocco, E. et al. AACR, 2015, Abstract 3103. 2. Scaltriti, M. et al. Proc Natl Acad Sci USA. 201, 108, 3761-6. 3. Horiuchi, D. et al. J Exp Med. 2012, 209, 679-96. Citation Format: Blake DG, MacKay C, Frame S, Zheleva D. CYC065, a novel CDK2/9 inhibitor: Molecular basis for clinical development in basal-like triple-negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-10.

Abstract B182: Molecular basis for clinical development of the novel CDK2/9 inhibitor CYC065 in oncology

CYC065 is a novel CDK2/9 inhibitor. Following completion of IND-enabling studies CYC065 has been cleared by the FDA for first-in-human clinical trials. This study aims to establish the mechanistic rationale and dosing schedule for targeting tumors that are either dependent on sustained expression of certain CDK9 transcriptional targets including Mcl-1 and MYC, or on activation of CDK2, e.g. by overexpression of cyclin E. CYC065 is efficacious in ALL and AML preclinical models, including MLL rearranged (MLLr) leukemia1. Mcl-1 is critical for survival of AML2, and MLLr AML are dependent on CDK9-driven expression of MLL target genes3. CYC065 is effective in cyclin E-overexpressing tumor models, such as uterine serous carcinoma4 and trastuzumab-resistant Her2+ breast cancer5. Pharmacologic CDK inhibition or CDK knockdown is synthetically lethal with overexpressed MYC in triple negative breast cancer (TNBC)6. A common feature of the basal-like subtype of TNBC is amplification or overexpression of cyclin E and MYC, suggesting potential utility for a CDK2/9 inhibitor. The efficacy and mechanism of action of CYC065 was analyzed in AML and basal-like TNBC cell panels by measuring the effects on cell proliferation, levels of key CDK9-dependent proteins, cell cycle distribution and apoptosis induction. We show that submicromolar CYC065 inhibits CDK9-dependent RNA Polymerase II phosphorylation, resulting in downregulation of target proteins and rapid induction of apoptosis. Sensitive tumor subtypes undergo rapid apoptosis after short pulse treatments at doses and durations predicted to be clinically achievable and well tolerated. However, significant apoptosis induction was not observed in non-malignant cells despite showing similar upstream effects. The data predicts a therapeutic window between cancer and normal proliferating cells and indicates the potential for stratification markers to enrich for sensitive patients. Combinability of anti-cancer agents is an important consideration for clinical development. In preclinical models Cyclacel9s first generation CDK inhibitor seliciclib combines effectively with the novel nucleoside analogue sapacitabine (or its active metabolite CNDAC). This combination is currently being evaluated in a phase 1 clinical trial (NCT00999401)7. We show that CYC065 combines effectively with CNDAC in a TNBC panel. Increased expression and activity of other anti-apoptotic Bcl-2-family members can counteract the pro-apoptotic effects of Mcl-1 downregulation by CYC065. This can be overcome by combined treatment with CYC065 and Bcl-2 inhibitors such as ABT-199, resulting in a highly synergistic combination. CYC065 has the potential to be efficacious in cancers that require sustained expression of CDK9-dependent transcripts or activation of CDK2. These include Mcl-1-dependent and MLLr leukemia, MYC-driven lymphoma, and MYC- or cyclin E-dependent breast or gynecological cancers. CYC065 can act synergistically with selected DNA damaging or targeted agents. Together this data suggests that CYC065 may be a promising novel anti-cancer agent. 1) Saladino et al. AACR 2015, Abs 1650 2) Glaser et al. Genes Dev. 2012, 26, 120-5 3) Dou & Hess. Int J Haematol. 2008, 87, 10-8 4) Cocco et al. AACR, 2015, Abs 3103 5) Scaltriti et al. Proc Natl Acad Sci USA. 201, 108, 3761-6 6) Horiuchi et al. J Exp Med. 2012, 209, 679-96 7) Shapiro et al. AACR, 2013, Abs LB-202 Citation Format: Craig MacKay, Sheelagh Frame, Chiara Saladino, Elizabeth Pohler, Daniella Zheleva, David G. Blake. Molecular basis for clinical development of the novel CDK2/9 inhibitor CYC065 in oncology. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B182.

Abstract 4178: The novel PLK1 inhibitor, CYC140: Identification of pharmacodynamic markers, sensitive target indications and potential combinations

Introduction: CYC140 is a selective and potent ATP-competitive inhibitor of PLK1, which has completed IND-enabling studies. The aim of this translational project was to inform the clinical development path of CYC140. Esophageal cancer was investigated as a potential target indication based on unmet medical need and the observation that PLK1 is frequently overexpressed in esophageal tumors and carries a poor prognosis. Experimental procedures: The anticancer activity of CYC140 was examined across multiple tumor types to identify sensitive target indications or tumor subsets. 6 h pulse exposure to CYC140 was used to determine sensitivity in a panel of over 250 cancer cell lines, including 15 esophageal cancer cell lines, using CellTiter Glo and resazurin-based assays. Candidate pharmacodynamic markers were examined in malignant and non-malignant cells. Drug combination testing was undertaken in several esophageal cell lines using approved and targeted agents. Solid tumor and leukemia xenograft models were performed to assess CYC140 dosing schedules and efficacy. Summary: Inhibition of PLK1 by CYC140 perturbs the entry into and exit from mitosis. In malignant cells, CYC140 treatment leads to the appearance of mitotic cells with monopolar spindles and a persistent increase in the proportion of cells in G2 and M phase, resulting in complete growth inhibition and induction of cell death. In non-malignant cells, the growth arrest is transient, and cells resume cycling once compound is removed. Short (6 h) pulse treatments of CYC140 maximise the difference in cellular response between malignant esophageal cell lines and cells of a non-malignant origin. In the esophageal cell line panel, CYC140 cellular IC50 correlates with induction of apoptosis. The effect of CYC140 on pharmacodynamic markers of PLK-1 activity such as phospho-nucleophosmin and phospho-histone H3 was characterized in malignant and non-malignant cells. Several promising combinations of CYC140 with targeted agents were identified, including EGFR inhibitors, HDAC inhibitors and PI3K pathway inhibitors, and CYC140 can also be combined with cytotoxic agents approved for use in esophageal cancer, such as cisplatin or irinotecan. CYC140 anti-tumor efficacy was demonstrated in solid tumor and leukemic xenograft models with responses including tumor regression and tumor-free cures. Conclusions: CYC140 is a promising anti-cancer agent with potent anti-proliferative activity and therapeutic potential in a variety of cancers, including esophageal cancer and acute leukemia. The mode of action of CYC140 is consistent with PLK1 inhibition and cell death is preferentially triggered in sensitive malignant cells. Suitable pharmacodynamic markers and several promising combinations have been identified that could assist clinical development of CYC140. Citation Format: Sylvie Moureau, Craig MacKay, Chiara Saladino, Elizabeth Pohler, Karin Kroboth, Jonathan Hollick, Daniella Zheleva, Sheelagh Frame, David Blake. The novel PLK1 inhibitor, CYC140: Identification of pharmacodynamic markers, sensitive target indications and potential combinations [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 4178. doi:10.1158/1538-7445.AM2017-4178

Abstract 1309: CYC065, a novel CDK2/9 inhibitor, is an effective inducer of cell death and synergizes with BCL2 and BET inhibitors in B-cell lymphoma, including double-hit lymphomas

Introduction: Double-hit lymphomas (DHL), defined by concurrent MYC and BCL2 rearrangements, have poor prognosis compared to standard-risk diffuse large B cell lymphomas (DLBCL). Current treatment regimens involve multiple chemotherapies, but do not specifically exploit these molecular features of the disease. Several studies have established that: (i) DLBCL show frequent overexpression of Mcl-1, an anti-apoptotic member of the Bcl-2 family (30% and 50% in the ABC and GCB subtypes, respectively), (ii) MYC-driven lymphomas are highly sensitive to depletion of Mcl-1, (iii) MYC overexpression and inhibition of CDK activity are synthetically lethal and (iv) resistance to BH3 mimetics targeting Bcl-2 can be conferred by upregulation of Mcl-1. CYC065 is a specific and potent CDK2/9 inhibitor, currently in a Phase 1 trial in patients with advanced cancer (NCT02552953). The mechanism of action of CYC065 involves rapid reduction of the levels of both Mcl-1 and MYC, suggesting a therapeutic rationale for investigating this agent in DLBCL. This preclinical study explored the single agent activity of CYC065 in B cell lymphoma, and its potential to combine with other molecularly targeted agents of interest, including venetoclax (ABT-199, a Bcl-2 inhibitor), and BET inhibitors. Methods: Single agent activity of CYC065 was explored using short pulse treatments (6-8 h) in B cell lymphoma cell lines. Viability and total cell number were assessed 24, 48 or 72 h following treatment. Levels of MYC, Bcl-2 and Mcl-1 were determined by Western blotting at baseline and following treatment with CYC065. Cell fate was examined by flow cytometry. CYC065 was combined with venetoclax or BET inhibitors ((+)-JQ-1, GSK525762 and OTX-015), which were administered concomitantly for a 6 h pulse or up to 72 h. Combination data were analyzed by the Chou & Talalay method. Results: The median IC 50 for CYC065 in 13 B cell lymphoma cell lines was 0.43 μM. No obvious difference was observed between ABC and GCB subtypes of DLBCL, and DHL DLBCL lines had similar IC 50 values to non-DHL DLBCL lines (median 0.47 μM vs 0.29 μM; p = 0.95). As expected from the target inhibitory profile, CYC065 caused a rapid decrease in the phosphorylation of S2 of the CTD of RNA polymerase II followed by downregulation of Mcl-1 and MYC, and rapid induction of apoptosis in sensitive cell lines. CYC065 had no impact on Bcl-2 levels. Combining CYC065 with venetoclax was highly synergistic in DHL lines (median CI values range from 0.1-0.8), and resulted in >90% cell death. The combination of CYC065 and BET inhibitors was also highly synergistic. Conclusions: CYC065 targets key oncogenic and survival pathways in DLBCL. CYC065 is a potent and effective inducer of cell death and combines synergistically with Bcl-2 or BET inhibitors in B cell lymphoma cell lines, including DHLs, which represent an unmet clinical need. Citation Format: Sheelagh M. Frame, Elizabeth Pohler, Craig MacKay, Daniella Zheleva, David Blake. CYC065, a novel CDK2/9 inhibitor, is an effective inducer of cell death and synergizes with BCL2 and BET inhibitors in B-cell lymphoma, including double-hit lymphomas. [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 1309.