Supplementary Materials and Methods Supplementary References Tables S1 to S3 and legends Figures S1 to S4 and legends

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Chemotherapy-induced peripheral neuropathy (CIPN) and chemotherapy-induced cognitive impairments (CICI) are common, often severe neurotoxic side effects of cancer treatment that greatly reduce quality of life of cancer patients and survivors. Currently, there are no Food and Drug Administration-approved agents for the prevention or curative treatment of CIPN or CICI. The dual leucine zipper kinase (DLK) is a key mediator of axonal degeneration that is localized to axons and coordinates the neuronal response to injury. We developed a novel brain-penetrant DLK inhibitor, IACS ' 8287, which demonstrates potent and highly selective inhibition of DLK in vitro and in vivo. Coadministration of IACS ' 8287 with the platinum derivative cisplatin prevents mechanical allodynia, loss of intraepidermal nerve fibers in the hind paws, cognitive deficits, and impairments in brain connectivity in mice, all without interfering with the antitumor activity of cisplatin. The protective effects of IACS ' 8287 are associated with preservation of mitochondrial function in dorsal root ganglion neurons and in brain synaptosomes. In addition, RNA sequencing analysis of dorsal root ganglia reveals modulation of genes involved in neuronal activity and markers for immune cell infiltration by DLK inhibition. These data indicate that CIPN and CICI require DLK signaling in mice, and DLK inhibitors could become an attractive treatment in the clinic when coadministered with cisplatin, and potentially other chemotherapeutic agents, to prevent neurotoxicities as a result of cancer treatment.

Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2) plays a role in receptor tyrosine kinase (RTK), neurofibromin-1 (NF-1), and Kirsten rat sarcoma virus (KRAS) mutant-driven cancers, as well as in RTK-mediated resistance, making the identification of small-molecule therapeutics that interfere with its function of high interest. Our quest to identify potent, orally bioavailable, and safe SHP2 inhibitors led to the discovery of a promising series of pyrazolopyrimidinones that displayed excellent potency but had a suboptimal in vivo pharmacokinetic (PK) profile. Hypothesis-driven scaffold optimization led us to a series of pyrazolopyrazines with excellent PK properties across species but a narrow human Ether-à-go-go-Related Gene (hERG) window. Subsequent optimization of properties led to the discovery of the pyrimidinone series, in which multiple members possessed excellent potency, optimal in vivo PK across species, and no off-target activities including no hERG liability up to 100 μM. Importantly, compound 30 (IACS-15414) potently suppressed the mitogen-activated protein kinase (MAPK) pathway signaling and tumor growth in RTK-activated and KRASmut xenograft models in vivo.

Tumor-associated macrophages (TAMs) have a significant presence in the tumor stroma across multiple human malignancies and are believed to be beneficial to tumor growth. Targeting CSF1R has been proposed as a potential therapy to reduce TAMs, especially the protumor, immune-suppressive M2 TAMs. Additionally, the high expression of CSF1R on tumor cells has been associated with poor survival in certain cancers, suggesting tumor dependency and therefore a potential therapeutic target. The CSF1-CSF1R signaling pathway modulates the production, differentiation, and function of TAMs; however, the discovery of selective CSF1R inhibitors devoid of type III kinase activity has proven to be challenging. We discovered a potent, highly selective, and orally bioavailable CSF1R inhibitor, IACS-9439 (1). Treatment with 1 led to a dose-dependent reduction in macrophages, promoted macrophage polarization toward the M1 phenotype, and led to tumor growth inhibition in MC38 and PANC02 syngeneic tumor models.

Inhibition of glutaminase-1 (GLS-1) hampers the proliferation of tumor cells reliant on glutamine. Known glutaminase inhibitors have potential limitations, and in vivo exposures are potentially limited due to poor physicochemical properties. We initiated a GLS-1 inhibitor discovery program focused on optimizing physicochemical and pharmacokinetic properties, and have developed a new selective inhibitor, compound 27 (IPN60090), which is currently in phase 1 clinical trials. Compound 27 attains high oral exposures in preclinical species, with strong in vivo target engagement, and should robustly inhibit glutaminase in humans.

Inhibiting glycolysis remains an aspirational approach for the treatment of cancer. We have previously identified a subset of cancers harbouring homozygous deletion of the glycolytic enzyme enolase (ENO1) that have exceptional sensitivity to inhibition of its redundant paralogue, ENO2, through a therapeutic strategy known as collateral lethality. Here, we show that a small-molecule enolase inhibitor, POMHEX, can selectively kill ENO1 -deleted glioma cells at low-nanomolar concentrations and eradicate intracranial orthotopic ENO1 -deleted tumours in mice at doses well-tolerated in non-human primates. Our data provide an in vivo proof of principle of the power of collateral lethality in precision oncology and demonstrate the utility of POMHEX for glycolysis inhibition with potential use across a range of therapeutic settings.

Abstract Src homology 2 domain-containing phosphatase (SHP2) is a phosphatase that mediates signaling downstream of multiple receptor tyrosine kinases (RTK) and is required for full activation of the MAPK pathway. SHP2 inhibition has demonstrated tumor growth inhibition in RTK-activated cancers in preclinical studies. The long-term effectiveness of tyrosine kinase inhibitors such as the EGFR inhibitor (EGFRi), osimertinib, in non–small cell lung cancer (NSCLC) is limited by acquired resistance. Multiple clinically identified mechanisms underlie resistance to osimertinib, including mutations in EGFR that preclude drug binding as well as EGFR-independent activation of the MAPK pathway through alternate RTK (RTK-bypass). It has also been noted that frequently a tumor from a single patient harbors more than one resistance mechanism, and the plasticity between multiple resistance mechanisms could restrict the effectiveness of therapies targeting a single node of the oncogenic signaling network. Here, we report the discovery of IACS-13909, a specific and potent allosteric inhibitor of SHP2, that suppresses signaling through the MAPK pathway. IACS-13909 potently impeded proliferation of tumors harboring a broad spectrum of activated RTKs as the oncogenic driver. In EGFR-mutant osimertinib-resistant NSCLC models with EGFR-dependent and EGFR-independent resistance mechanisms, IACS-13909, administered as a single agent or in combination with osimertinib, potently suppressed tumor cell proliferation in vitro and caused tumor regression in vivo. Together, our findings provide preclinical evidence for using a SHP2 inhibitor as a therapeutic strategy in acquired EGFRi-resistant NSCLC. Significance: These findings highlight the discovery of IACS-13909 as a potent, selective inhibitor of SHP2 with drug-like properties, and targeting SHP2 may serve as a therapeutic strategy to overcome tumor resistance to osimertinib.

Increased expression of IDO1 is believed to create a tumor microenvironment that is immunosuppressive. In the course of our research directed at identifying potent and selective inhibitors of IDO1, we identified a class of compounds that inhibited IDO1 activity in a cellular context, but not in isolated enzymatic assays. We have conducted detailed mechanistic studies and shown that these molecules inhibit IDO1 by binding to the apo-enzyme, thus preventing the incorporation of the heme-cofactor into the active site of the holo-enzyme. Through an extensive medicinal chemistry campaign, we optimized a series of orally bioavailable, highly potent and selective inhibitors of IDO1 that possess excellent pharmacological properties. For several lead molecules, pharmacokinetic (PK) - pharmacodynamic (PD) relationships were established in whole blood and SKOV3 xenograft assays. The inhibition of IDO1 in a human whole-blood assay correlated well with the suppression of tumor kynurenine (KYN) that was observed in SKOV3 xenografts. At plasma concentrations of 3 µM, IACS-9779 supressed tumor KYN levels by 90%. IACS-9779 was well tolerated with excellent in vivo PK properties across multiple preclinical species, and a human PK prediction consistent with a low daily dose needed for full suppression of KYN production via IDO1. Note: This abstract was not presented at the meeting. Citation Format: Faika Mseeh, Matthew M. Hamilton, Joseph R. Marszalek, Norma E. Rogers, Connor A. Parker, Simon S. Yu, Zhen Liu, Naphtali J. Reyna, Timothy McAfoos, Brett W. Virgin-Downey, Paul G. Leonard, Jason B. Cross, Ningping Feng, Angela L. Harris, Andy M. Zuniga, Keith Mikule, Martin Tremblay, Yongying Jiang, Mikhila Mahendra, Jihai Pang, Qi Wu, Quanyun Xu, Timothy P. Heffernan, Philip Jones, Richard T. Lewis. IACS-9779, a development candidate that inhibits 2,3-dioxygenase (IDO) activity by blocking heme incorporation into IDO apoenzyme [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 3277.

Abstract Osimertinib, a third generation EGFR inhibitor, is a front-line therapy for EGFR mutated non-small lung cancer (NSCLC). The long-term effectiveness of osimertinib is limited by acquired resistance. Clinically identified resistance mechanisms include EGFR-dependent mechanisms such as mutations on EGFR that preclude drug binding, and EGFR-independent activation of the MAPK pathway, for instance via activation of alternate RTKs. It has also been noted that frequently a tumor from a single patient harbors more than one resistance mechanism, and the plasticity between the multiple resistance mechanisms will restrict the effectiveness of therapies targeting a single node of the oncogenic signaling network. SHP2 (Src homology 2 domain-containing phosphatase) is a phosphatase that mediates the signaling of multiple RTKs and is required for full activation of the MAPK pathway. Here we report IACS-13909 - a specific and potent allosteric inhibitor of SHP2 - suppresses the signaling of RTK/MAPK pathway. IACS-13909 potently impedes the proliferation of tumors with a broad spectrum of RTKs as the oncogenic driver. Importantly, in NSCLC models with acquired resistance to osimertinib, IACS-13909 administered as a single agent or in combination with osimertinib potently reduces tumor cell proliferation in vitro and in vivo. Together, our findings provide preclinical evidence for using a SHP2 inhibitor as a therapeutic strategy in acquired EGFR inhibitor-resistant NSCLC. Currently, a compound that potently inhibits SHP2 has been selected as the clinical development candidate and is undergoing IND-enabling studies with a projected first-in-human target of early 2020. Citation Format: Yuting Sun, Brooke A Meyers, Sarah B Johnson, Angela L Harris, Barbara Czako, Jason B Cross, Paul G Leonard, Faika Mseeh, Maria E Di Francesco, Connor A Parker, Qi Wu, Christopher A Bristow, Jason P Burke, Caroline C Carrillo, Christopher L Carroll, Qing Chang, Ningping Feng, Sonal Gera, Gao Guang, Justin Kwang-Lay Huang, Yongying Jiang, Zhijun Kang, Jeffrey J Kovacs, Xiaoyan Ma, Pijus K Mandal, Timothy McAfoos, Robert A Mullinax, Michael D Peoples, Vandhana Ramamoorthy, Sahil Seth, Erika Suzuki, Christopher Conrad Williams, Simon S Yu, Andy M Zuniga, Giulio F Draetta, Joseph R Marszalek, Timothy P Heffernan, Nancy E Kohl, Philip Jones. Discovery of IACS-13909, an allosteric SHP2 inhibitor that overcomes multiple mechanisms underlying osimertinib resistance [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C036. doi:10.1158/1535-7163.TARG-19-C036

Ibrutinib is highly efficacious and used at 420 mg/d for treatment of chronic lymphocytic leukemia (CLL). We previously demonstrated a decline in Bruton's tyrosine kinase (BTK) protein levels in CLL cells after 1 cycle of ibrutinib, suggesting ibrutinib dose could be lowered after the first cycle without loss of biological effect. To test this postulate, a pilot study (NCT02801578) was designed to systematically reduce ibrutinib dosing within the same patient with CLL over the course of three 28-day cycles. After an initial cycle of 420 mg/d, the dose was reduced to 280 mg/d in cycle 2, and then to 140 mg/d in cycle 3. Eleven patients began study treatment, and 9 completed the 3 cycles. Plasma and intracellular pharmacokinetics (PK), BTK occupancy, and pharmacodynamic (PD) response at different doses of ibrutinib were compared. Plasma and intracellular levels of ibrutinib were dose-dependent, and even the lowest dose was sufficient to occupy, on average, more than 95% of BTK protein. In concert, BTK downstream signaling inhibition was maintained with 140 mg/d ibrutinib in cycle 3, and there were comparable reductions in total and phospho-BTK (Tyr223) protein levels across 3 cycles. Reductions of plasma chemokine CCL3 and CCL4 levels, considered to be biomarkers of ibrutinib response, were similar during the 3 cycles. These PK/PD data demonstrate that after 1 cycle of ibrutinib at the standard 420 mg/d dose, the dose can be reduced without losing biological activity. Clinical efficacy of lower doses needs to be systematically evaluated. Such dose reductions would lower drug cost, lessen untoward toxicity, and facilitate rationale-based combinations. This trial was registered at www.clinicaltrials.gov as #NCT02801578.

ABSTRACT Inhibiting glycolysis remains an aspirational approach for the treatment of cancer. We recently demonstrated that SF2312, a natural product phosphonate antibiotic, is a potent inhibitor of the glycolytic enzyme Enolase with potential utility for the collateral lethality-based treatment of Enolase-deficient glioblastoma (GBM). However, phosphonates are anionic at physiological pH, limiting cell and tissue permeability. Here, we show that addition of pivaloyloxymethyl (POM) groups to SF2312 (POMSF) dramatically increases potency, leading to inhibition of glycolysis and killing of ENO1 -deleted glioma cells in the low nM range. But the utility of POMSF in vivo is dose-limited by severe hemolytic anemia. A derivative, POMHEX, shows equipotency to POMSF without inducing hemolytic anemia. POMHEX can eradicate intracranial orthotopic ENO1 -deleted tumors, despite sub-optimal pharmacokinetic properties. Taken together, our data provide in vivo proof-of-principal for collateral lethality in precision oncology and showcase POMHEX as a useful molecule for the study of glycolysis in cancer metabolism.

Abstract Indoleamine 2,3-dioxygenase (IDO1 and IDO2) and tryptophan dioxygenase (TDO) are heme-containing enzymes that mediate the rate limiting step in the oxidative degradation of L-tryptophan (L-TRP) to kynurenine (KYN) metabolites. Tryptophan catabolism through the KYN metabolic pathway is now recognized as one of many mechanisms involved in tumor cell evasion of the immune surveillance system. Inhibition of the KYN pathway in the tumor microenvironment can lead to improved immune response and tumor growth suppression. Recently, clinical proof of concept of this mechanism has been demonstrated using an Indoleamine 2,3-dioxygenase (IDO1) inhibitor in combination with a PD-1 antagonist in a variety of tumor contexts. Consideration of known low molecular weight heme-co-ordinating ligands identified from the PDB, in conjunction with a virtual screen performed in-silico identified a number of potentially interesting starting points for medicinal chemistry development. Identification of an attractive indazole fragment as a starting point, and expansion into alternative bicyclic cores, resulted in the discovery of a family of imidazopyridines as potent human IDO1 inhibitors with >200 fold selectivity against TDO. Utilizing a structure-based design approach allowed rapid lead optimization that resulted in the identification of IACS-8968. Crystallography studies were conducted, and binding of IACS-8968 to the heme domain of the human IDO1 was confirmed. The homochiral imidazopyridine IACS-8968 displayed cellular IC50= 29 nM in a HeLa cell line expressing human IDO1 and IC50= 21 nM in a PANC02 mouse cell line expressing the murine IDO1 enzyme, showed satisfactory selectivity margin (> 150 fold) versus its CYP450 inhibition profile and good oral bioavailability across species. PK/PD experiments indicated that, at equivalent exposure, IACS-8968 (sodium salt) and epacadostat decreased tumor KYN at comparable levels in CT26 syngeneic mouse model. Citation Format: Alessia Petrocchi, Naphtali J. Reyna, Faika Mseeh, Connor A. Parker, Simon Yu, Quanyun Xu, Ningping Feng, Paul Leonard, Norma Rogers, Jason B. Cross, Angela L. Harris, Yongying Jiang, Tin Oo Khor, Mikhila G. Mahendra, Jihai Pang, Qi Wu, Andy M. Zuniga, Timothy McAfoos, Timothy McAfoos, Matthew M. Hamilton, Joe R. Marszalek, Keith Mikule, Paul Vancutsem, Keith Wilcoxen, Martin Tremblay, Philip Jones, Richard T. Lewis. Discovery of an imidazopyridine series of potent human IDO1 inhibitors with robust target engagement in a preclinical tumor model [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 LB-071.

High-content, image-based screens enable the identification of compounds that induce cellular responses similar to those of known drugs but through different chemical structures or targets. A central challenge in designing phenotypic screens is choosing suitable imaging biomarkers. Here we present a method for systematically identifying optimal reporter cell lines for annotating compound libraries (ORACLs), whose phenotypic profiles most accurately classify a training set of known drugs. We generate a library of fluorescently tagged reporter cell lines, and let analytical criteria determine which among them--the ORACL--best classifies compounds into multiple, diverse drug classes. We demonstrate that an ORACL can functionally annotate large compound libraries across diverse drug classes in a single-pass screen and confirm high prediction accuracy by means of orthogonal, secondary validation assays. Our approach will increase the efficiency, scale and accuracy of phenotypic screens by maximizing their discriminatory power.