Supplementary Figures S1-S6 and legends, and Tables S1, S2, S5, S6, S7.

Gene sets significantly upregulated upon USP7 inhibitor treatment of H526 cells.

Genes whose expression is significantly altered upon USP7 inhibitor treatment of H526 cells.

Abstract Hematopoietic progenitor kinase 1 (HPK1) is an intracellular protein kinase that negatively regulates T cell signaling and proliferation. Upon T cell receptor (TCR) activation, active HPK1 phosphorylates the adaptor protein SLP76 in the TCR complex, recruiting the negative regulator 14-3-3 and targeting components of the TCR signaling complex for degradation. HPK1 thus limits the TCR signaling important for mounting an effective immune response against tumor cells. We are employing structure-guided drug design to develop potent small-molecule inhibitors of HPK1. Our compounds potently inhibit HPK1 in biochemical assays, reduce levels of phosphorylated SLP76 and concomitantly increase IL-2 production by Jurkat T cells. Importantly, our HPK1 inhibitors enhance cytokine production by human and mouse primary T cells above that observed with TCR activation alone. Treatment of mice with orally available HPK1 inhibitors results in increased activation of antigen-specific CD8+ T cells in vivo and decreased tumor growth as single agent and in combination with clinically relevant checkpoint inhibitor antibodies. Our work confirms the importance of HPK1 for T cell function and supports HPK1 as a promising next generation immuno-oncology target. Citation Format: George Katibah, Yamini Ohol, Cyril Bucher, Lavanya Adusumilli, Deepika Kaveri, Omar Robles, Michael Sun, Cynthia Cho, Heather Milestone, Rachel Ames, Scott Jacobson, Dan Nebalasca, Justy Gomez- Guagua, Jerick Sanchez, Molly Grandcolas, Steve Wong, Martin Brovarney, Chandru Ramana, Thant Zaw, Lan Nguyen, Parcharee Tivitmahaisoon, Andrew Ng, Anqi Ma, Blanca Gomez, Michelle Ko, Paul Leger, Jeffrey Jackson, Grant Shibuya, Anton Shakhmin, Delia Bradford, Mengshu Xu, Mikhail Zibinsky, Daniel Poon, David Wustrow, Paul Kassner, Dirk Brockstedt. Development of small-molecule HPK1 inhibitors to unleash tumor-specific T cell responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1646.

USP7 is a promising target for cancer therapy as its inhibition is expected to decrease function of oncogenes, increase tumor suppressor function, and enhance immune function. Using a structure-based drug design strategy, a new class of reversible USP7 inhibitors has been identified that is highly potent in biochemical and cellular assays and extremely selective for USP7 over other deubiquitinases. The succinimide was identified as a key potency-driving motif, forming two strong hydrogen bonds to the allosteric pocket of USP7. Redesign of an initial benzofuran-amide scaffold yielded a simplified ether series of inhibitors, utilizing acyclic conformational control to achieve proper amine placement. Further improvements were realized upon replacing the ether-linked amines with carbon-linked morpholines, a modification motivated by free energy perturbation (FEP+) calculations. This led to the discovery of compound 41, a highly potent, selective, and orally bioavailable USP7 inhibitor. In xenograft studies, compound 41 demonstrated tumor growth inhibition in both p53 wildtype and p53 mutant cancer cell lines, demonstrating that USP7 inhibitors can suppress tumor growth through multiple different pathways.

Abstract The deubiquitinase USP7 regulates the levels of multiple proteins with roles in cancer progression and immune response. Thus, USP7 inhibition may decrease oncogene function, increase tumor suppressor function, and sensitize tumors to DNA-damaging agents. We have discovered a novel chemical series that potently and selectively inhibits USP7 in biochemical and cellular assays. Our inhibitors reduce the viability of multiple TP53 wild-type cell lines, including several hematologic cancer and MYCN-amplified neuroblastoma cell lines, as well as a subset of TP53-mutant cell lines in vitro. Our work suggests that USP7 inhibitors upregulate transcription of genes normally silenced by the epigenetic repressor complex, polycomb repressive complex 2 (PRC2), and potentiate the activity of PIM and PI3K inhibitors as well as DNA-damaging agents. Furthermore, oral administration of USP7 inhibitors inhibits MM.1S (multiple myeloma; TP53 wild type) and H526 (small cell lung cancer; TP53 mutant) tumor growth in vivo. Our work confirms that USP7 is a promising, pharmacologically tractable target for the treatment of cancer.

USP7 is a deubiquitinase that regulates the levels of multiple downstream targets with roles in cancer progression and immune response. Inhibitors of USP7 may thus decrease oncogene function, increase tumor suppressor function, enhance immune function and sensitize tumor cells to DNA damaging agents. We have discovered a novel chemical series that potently and selectively inhibits USP7 in biochemical and cellular assays. Our inhibitors reduce the viability of multiple p53-wild type cell lines, including several blood cancer and MYCN-amplified neuroblastoma cell lines, as well as a subset of p53-mutant tumor cell lines in vitro. Further, oral administration of our USP7 inhibitors inhibits MM.1S (multiple myeloma; p53-wild type) and H526 (small cell lung cancer; p53-mutant) tumor growth in vivo. Our work confirms that USP7 is a pharmacologically tractable target and future studies will aim to further understand the mechanism of action of USP7 inhibitors in p53-mutant cancers. Citation Format: Yamini M. Ohol, Michael Sun, Paul Leger, Dennis Hu, Berenger Biannic, Payal Rana, Cynthia Cho, Scott Jacobson, Steve Wong, Jerick Sanchez, Xinping Han, Kyle Young, Akinori Okano, Jack Maung, Gene Cutler, Nick Shah, Lavanya Adusumilli, Deepika Kaveri, Oezcan Talay, Deepa Pookot, Betty Abraham, Delia Bradford, Nathan Kozon, Christophe Colas, Andrea Kim, Jacob Schwarz, David Wustrow, Dirk Brockstedt, Paul Kassner. Discovery of potent and selective inhibitors of USP7 with anti-tumor activity in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4441.

USP7 is a deubiquitinase (DUB) that has attracted much attention recently due to its multiple roles in promoting cancer progression. By removal of ubiquitin from protein substrates, USP7 stabilizes oncogenes such as MDM2 and Myc, destabilizes and inactivates the key tumor suppressors p53 and PTEN, and imparts resistance to DNA-damaging chemotherapy by enhancing DNA repair responses. USP7 plays an important role in suppression of immune responses in the tumor microenvironment by stabilizing the transcription factor FOXP3 and thereby enhancing the suppressive function of regulatory T cells. Thus, inhibition of USP7 is an appealing therapeutic strategy because it has the potential to impact important oncology targets such as transcription factors that have been widely viewed as undruggable. We employed structure-based and other medicinal chemistry techniques to enable the design of potent and selective USP7 inhibitors. Using a high-throughput assay of DUB activity employing rhodamine-labeled ubiquitin, we optimized several series of reversible USP7 inhibitors to sub-100 pM potency and selectivity of >10,000-fold over all other DUBs. Cellular activity was demonstrated using a luciferase reporter gene assay of p53 activation, revealing compounds with EC50 values ranging down to 20 nM. To assess the role of USP7 inhibition in enhancement of immune responses, we determined relief of suppression of effector T cells in vitro. Effector T cells (CD8+) were co-cultured with regulatory T cells (CD4+ FOXP3+) and antigen-presenting cells for 4 days, after which CD8+ cell proliferation was determined by flow cytometry. Treatment with USP7 inhibitors during co-culture resulted in relief of regulatory T cell suppression of CD8+ cell proliferation. In vivo enhancement of immune responses was assessed in rodent models of inflammation and tumor growth. Direct effects on tumor cell growth and viability were explored by profiling cytotoxicity of USP7 inhibitors as single agents and in combination with chemotherapeutic agents in a broad range of cancer cell lines. In preparation for future clinical development, compounds were modified to obtain desirable in vitro and in vivo ADME and toxicity profiles. Following extensive pre-clinical optimization, we have in hand orally bioavailable compounds with high permeability, low clearance, and minimal off-target activity. Citation Format: Betty Abraham, Lavanya Adusumilli, Berenger Biannic, Delia Bradford, Martin Brovarney, David Chian, Gene Cutler, Xinping Han, Dennis Hu, Scott Jacobson, Sherra Johnson, Paul Kassner, Deepika Kaveri, John Ketcham, Andrea Kim, Paul Leger, Lisa Marshall, Sachie Marubayashi, Jack Maung, Jenny McKinnell, Cesar Meleza, Yamini Ohol, Akinori Okano, Leanne Peiser, Deepa Pookot, Payal Rana, Jacob Schwarz, Nick Shah, Grant Shibuya, Michael Sun, Silpa Suthram, Oezcan Talay, Angela Wadsworth, David Wustrow, Kyle Young, Andrew Napper. Discovery and optimization of potent and selective inhibitors of USP7 to enhance anti-tumor immunity and target tumor growth [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 2915.