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Acetylation of histones and additional nuclear proteins is a key mechanism in the regulation of gene expression. Aberrant acetylation has been linked to a wide range of diseases including cancer, inflammation, and neurodevelopmental disorders. Histone acetylation is introduced by histone acetyltransferases complexes (HATs), where substrate specificity is dramatically enhanced by scaffolding proteins that activate and target them to specific chromatin sites. A protein of interest with both epigenetic acetyl reader and scaffolding function is the protein Bromodomain and PHD finger-containing protein 1 (BRPF1). The Protein contains domains of two plant homeodomain (PHD) fingers separated by a zinc knuckle (PZP domain), a bromodomain, and a proline-tryptophan-tryptophan-proline (PWWP) Tudor domain. Native BRPF1 complexes can contain either MOZ/MORF or HBO1 as the catalytic MYST-family acetyltransferase subunit and the stable complex with Moz-Tif2 is known to lead to the development of human acute myeloid leukemia (AML). Given this, we chose to pursue the development of BRPF1 degraders to probe cancer disease biology especially in AML, where new therapies are required to overcome several unmet needs such as less-toxic treatments and relapsed/refractory disease paradigms. Here we present the design and synthesis of a range of BRPF1 degraders in highly desirable physicochemical space utilizing in-silico modeling. The prepared degraders which utilize multiple E3 ligases were then screened against cell lines harboring Mixed-lineage leukemia (MLL) translocations specifically the THP-1 cell line. In addition to this, we investigated the ability of the compounds to effectively degrade the target and suitability of our degraders for potential in-vivo exposure through a panel of routine ADMET assays. Citation Format: Daniel Joseph Glynn, Rosie Crampton, Thomas Pesnot, Andrew Scott, Anne-Chloe Nassoy, Ralph Kirk, Daniele Narducci, Gary Nelson, Lynette Ongiri, Habiba Begum, Vincent Rao, Matilda Bingham, Rhoanne McPherson, Darryl Turner. The development of BRPF1 degraders as a potential treatment for acute myeloid leukemia [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 419.

Poly(ADP-ribose) glycohydrolase (PARG) hydrolyzes poly(ADP-ribose) (PAR) chains that are polymerized by PARP enzymes, completing the PAR cycle. Small molecule inhibitors of PARG result in a dose dependent increase in cellular PAR after DNA damage. Here we demonstrate that depletion of XRCC1, a scaffolding protein with an essential role in base-excision repair (BER), sensitizes cancer cells to PARG inhibition. XRCC1 deficient cells exhibit increased nuclear PAR foci in response to PARG inhibition even in the absence of DNA damaging agents. Inhibition of PARP1 with RNAi or small molecule inhibitors rescues cell growth inhibition and reduces the amount of cellular PAR accumulation in PARG inhibitor treated cells. This indicates that the cellular growth inhibition is dependent upon cellular PAR levels, demonstrating selectivity of the small molecule inhibitors for PARG. We hypothesized that inhibition of PAR hydrolysis could result in depletion of cellular NAD as this could prevent recycling of PAR to NAD. Consistent with this hypothesis, PARG inhibition enhanced NAD depletion after treatment of cells with the DNA damaging agent methyl methanesulfonate (MMS). Live cell imaging of XRCC1 depleted cells treated with a PARG inhibitor revealed that cells have large membrane protrusions, similar to the morphology of cells that have been treated with a NAMPT inhibitor, which results in depletion of cellular NAD. Furthermore, addition of the NAD precursor, nicotinamide mononucleotide (NMN) rescued proliferation of PARG inhibited cells. Taken together, these data support a hypothesis in which PARG inhibitors are cytotoxic to sensitive cancer cells via depletion of NAD, ultimately starving the cell of ATP. Thus, PARG inhibition is a novel strategy for exploiting synthetic lethality in cancer cells. The defects that sensitize cancer cells to PARG inhibition are distinct from those that sensitize to PARP inhibitors, namely defects in homology directed repair. Approximately 15% of breast cancer samples exhibit low or no XRCC1 by IHC. A subset (approximately 35%) of the XRCC1 low patient samples also have defects in BRCA1, suggesting that the majority of XRCC1 low tumors may not be responsive to PARP inhibitors. Small molecule PARG inhibitors are currently being evaluated for efficacy in XRCC1 low xenograft models. Citation Format: Leenus Martin, Tzuling Cheng, Dominic I. James, Habiba Begum, Kate M. Smith, Allan Jordan, Ian Waddell, Kedar Vaidya, Marcus Fischer, Bing Yao, Jason Drummond, Leah Cleary, Ruben Martinez, James Sutton, Nandini Ravindran, James Joseph, Eleni Venetsanakos, Michael Dillon, Jeffrey H. Hager, Lisa D. Belmont. PARG inhibitors exhibit synthetic lethality with XRCC1 deficiency and a cellular mechanism of action that is distinct from PARP inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1943.

Activating gene fusions in the RET receptor tyrosine kinase have been found to drive 1-2% of lung adenocarcinomas and therefore offer an attractive target for targeted therapy. Whilst non-selective tyrosine kinase inhibitors with RET activity are efficacious in this setting, their use is generally limited by dose limiting toxicity associated with their more potent activity versus other targets, specifically KDR (VEGFR2) in the case of cabozantinib and vandetanib. Given this limitation, there is considerable interest in developing more selective inhibitors of RET kinase. Tyrosine kinase inhibitors are prone to early clinical failure due to mutations in the kinase ATPase binding domain, which render the kinase catalytically active but no longer sensitive to drug treatment. Such mutations often occur in the so-called “gatekeeper” region and in this specific case, resistance is predicted to arise from a Val-Met or Val-Leu mutation at residue 804. Through a combination of computational methods, structural biology and drug design, we have identified and further optimized a series of inhibitors of the V804M mutant RET kinase which show sub-micromolar cellular activity in cells driven by V804M RET. Moreover, these agents show excellent selectivity against the wtRET kinase and KDR. As such, these agents may offer valuable start-points for second-generation RET inhibitors for use in patents who relapse after treatment with first generation selective RET inhibitors. Citation Format: Allan M. Jordan, Rebecca Newton, Bohdan Waszkowycz, Richard Bayliss, Habiba Begum, Daniel Burschowsky, Aude Echalier, Samantha Hitchin, Colin Hutton, Shaun Johns, Stuart Jones, Li-Ying Lin, Mark Richards, Chitra Seewooruthun, Alex Stowell, Ian Waddell, Mandy Watson, Donald Ogilvie. Delivering selective and cell-active inhibitors of V804M mutant RET kinase through structure-guided drug discovery [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 3236. doi:10.1158/1538-7445.AM2017-3236

Background: The aim of this CRUK-MI Drug Discovery project is to deliver a RET-selective inhibitor for the treatment of cancers with RET activating mutations, which include 1-2% of lung adenocarcinomas and medullary thyroid cancers (MTC). Recent data supports the hypothesis that the efficacy of vandetanib and cabozantinib, clinically approved multi-kinase inhibitors, is limited by toxicities associated with potent activity against KDR. Therefore, a RET-selective inhibitor would represent a best-in-class agent for the treatment of these cancers. Methods: We have established a robust screening cascade to develop a potent, selective RET inhibitor and developed several in vivo models to evaluate compound PKPD and antitumor efficacy. Tumor growth inhibition and PKPD studies were carried out in BaF3 mouse allograft models overexpressing KIF5B-RET or RETV804M and other disease relevant models, including an MTC xenograft (MZ-CRC-1), a KIF5B-RET lung cancer patient derived xenograft (PDX) model (CTG-0838, Champions Oncology) and a lung cancer control xenograft (Calu-6). Results: Two orally bioavailable compounds displaying nanomolar RET potency and >10 fold selectivity over KDR in cellular assays were selected from the lead series and further evaluated in our in vivo PD and efficacy models. Both compounds demonstrated efficacy in the BaF3 KIF5B-RET driven model (71% and 103% tumor growth inhibition (TGI), respectively), accompanied by reduced levels of pRET in the tumor tissue. Following further lead optimisation; a compound displaying an improved DMPK profile and additional nanomolar potency versus the gatekeeper mutation (RETV804M) was identified and accelerated through our DMPK/in vivo cascade. We consider this additional activity versus RETV804M beneficial since mutations at the gatekeeper residue in other tyrosine kinases (e.g. EGFR) have been shown to mediate acquired drug resistance in the clinic. This compound demonstrated significant TGI of 58% and 82% respectively in the BaF3 KIF5B-RET and BaF3 RETV804M allograft models. Moreover, tumor growth in the lung cancer PDX model was strongly inhibited (95% TGI) and tumor regression induced in the MTC xenograft model (109% TGI). As expected, this potent and selective RET inhibitor was not active in the Calu-6 model, which is sensitive to KDR inhibition, whereas vandetanib, a potent KDR inhibitor, significantly inhibited tumor growth (84% TGI). Additional in vitro and in vivo DMPK analyses further support the nomination of this compound as a preclinical candidate. Conclusions: The identification of selective RET inhibitors with significant in vivo activity and minimal toxicity may overcome the limitations of the currently available clinical compounds. We have made considerable progress towards this goal and show here the compelling data supporting our nomination of a preclinical development compound. Citation Format: Mandy Watson, Helen Small, Ben Acton, Habiba Begum, Samantha Hitchin, Allan Jordan, Paul Kelly, Rebecca Newton, Ian Waddell, Gina Paris, Donald Ogilvie. A potent and selective RET inhibitor with efficacy in RET-driven mouse models of medullary thyroid carcinoma and lung adenocarcinoma [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 2092. doi:10.1158/1538-7445.AM2017-2092