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

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.

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

Highlights • Segmental neurofibromatosis with MPNST showing multiple recurrences has not been reported in literature. • Studies were not conducted regarding the survival rate of such patients due to its extreme low incidence. • Being considered as a highly aggressive sarcoma, our patient with MPNST survived for a long period without distant metastasis. • Maintenance of old records helped to carry out review workup effectively., Introduction We present a rare case of Malignant Peripheral Nerve Sheath Tumour (MPNST) of the upper limb, which was excised thirteen times in thirteen years and ultimately ended in above elbow amputation. Presenting complaint and investigations A 48 year old female presented initially with a localised swelling of 2 cms diameter in the front of the left elbow in 2007, which was excised. It recurred repeatedly and was excised. In the earlier presentations, the swellings were firm, mobile and not fixed to bone. In the last stage alone, bone fixity was identified. All the fourteen surgeries were performed by the primary author from 2007 to 2020, as the patient was particular. The main clinical diagnoses had been neurofibroma and fibrosarcoma. There was no evidence of distant metastasis all these years. She did not respond to radiation or chemotherapy. Initially it was single, but later multiple. She had no clinical features of Neurofibromatosis 1 (NF1) or any family history. As the history progressed, the swellings became muscle deep and later encircled the radial nerve. The radial nerve was salvaged on three occasions. On the last three occasions, the tumour had to be shaved off from the humerus. The final amputation specimen showed a single tumour infiltrating the humerus and x-ray revealed bone destruction and tumour calcification. Final diagnosis was aided by immunohistochemistry (IHC) and cytogenetic study (FISH). Conclusion The case is presented for the rarity of the presentation and the trust and dependence of the patient on her personal surgeon.

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.

Recruitment of suppressive CD4+ FOXP3+ regulatory T cells (Treg) to the tumor microenvironment (TME) has the potential to weaken the antitumor response in patients receiving treatment with immuno-oncology (IO) agents. Human Treg express CCR4 and can be recruited to the TME through the CC chemokine ligands CCL17 and CCL22. In some cancers, Treg accumulation correlates with poor patient prognosis. Preclinical data suggests that preventing the recruitment of Treg and increasing the population of activated effector T cells (Teff) in the TME can potentiate antitumor immune responses. We developed a novel series of potent, orally bioavailable small molecule antagonists of CCR4. From this series, several compounds exhibited high potency in distinct functional assays in addition to good in vitro and in vivo ADME properties. The design, synthesis, and SAR of this series and confirmation of its in vivo activity are reported.

Arterial blood gas (ABG) analysis assesses the adequacy of ventilation, oxygenation, and the acid-base status of the body by measuring the levels of pH, oxygen, carbon dioxide, and bicarbonate in arterial blood. Interpretation of ABG results, which can play a major role in diagnosis and treatment of patients with pulmonary and other critical conditions, can sometimes be difficult. This article focuses on basic ABG analysis and interpretation, discusses the combinations of imbalances that may occur, and reviews the compensatory mechanisms that arise as a result.

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.

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

Recruitment of suppressive CD4

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.