Supplementary Figure and Table Legends from Multiple Roles for the Receptor Tyrosine Kinase Axl in Tumor Formation

Supplementary Figures 1-4, Table 1 from Multiple Roles for the Receptor Tyrosine Kinase Axl in Tumor Formation

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

Conclusions• CDK11 was identified from an in vivo functional genomics screen as a novel therapeutic target in PCAD patients.• Pharmacological inhibition with potent and selective tool compounds revealed a disconnect with genetic validation data.• Minimal tumor growth inhibition was observed in mouse efficacy studies as exposures were limited due to toxicity.• Taken together, our multidisciplinary approach to evaluate genetic and pharmacological inhibition of CDK11 suggests that while there is a dependency of a subset of tumor cells on CDK11, systemic inhibition of CDK11 in a whole organism is not tolerated, thereby limiting the therapeutic potential.

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.

Indoleamine 2,3-dioxygenase 1 (IDO1), a heme-containing enzyme that mediates the rate-limiting step in the metabolism of l-tryptophan to kynurenine, has been widely explored as a potential immunotherapeutic target in oncology. We developed a class of inhibitors with a conformationally constrained bicyclo[3.1.0]hexane core. These potently inhibited IDO1 in a cellular context by binding to the apoenzyme, as elucidated by biochemical characterization and X-ray crystallography. A SKOV3 tumor model was instrumental in differentiating compounds, leading to the identification of IACS-9779 (62) and IACS-70465 (71). IACS-70465 has excellent cellular potency, a robust pharmacodynamic response, and in a human whole blood assay was more potent than linrodostat (BMS-986205). IACS-9779 with a predicted human efficacious once daily dose below 1 mg/kg to sustain >90% inhibition of IDO1 displayed an acceptable safety margin in rodent toxicology and dog cardiovascular studies to support advancement into preclinical safety evaluation for human development.

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.

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.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

CONTEXT:Chronic inflammation is a new catch phrase for the explanation of all chronic degenerative diseases, from asthma, arthritis, heart disease, auto-immune disease, and irritable bowel disease to cancer. Occult infections from oncovirus, bacterial, and fungal infections as well as from lesser known parasitic infections are driving forces in the cellular evolution and degeneration of cancer cells. An approach using currently available medications that target both fungal and parasitic metabolism appears to interfere with the metabolic synergy that is associated with tumor growth and aggressiveness.OBJECTIVE:The review examined whether antiparasitic and antifungal medications that interfere with the metabolism of cancers, can be useful in cancer therapy by treating cancer as an infectious disease and as a metabolic parasite.DESIGN:The research team searched the National Center for Biotechnology Information (NCBI) PubMed database databases, using different keyword combinations, including repurposed drug, antifungal, antiparasitic, cancer, parasite, anti-cancer repurposed.SETTING:Prevention and Healing, St Louis, Mo, USA.RESULTS:The literature search identified a number of studies, including in vitro, in vivo and clinical, which support the use of antifungal and antiparasitic medication in the treatment of cancer. In the clinical area, the authors observed benefit from the use of antifungal and antiparasitic medication in the treatment of a variety of cancer cases.CONCLUSIONS:Due to the complexity of the behavior and biology of cells, scientists' primary focus should be on detection and elimination of sources of inflammation. Antiparasitic medications, and also antiviral, antibiotic, and antifungal medications should be thought of as underrecognized, underappreciated, and forgotten medications that can be part of cancer therapy. The information offered in this review suggests scientists should think of cancer not only as a metabolic disease but also as a metabolic parasite and should consider using antiparasitic medications under a new understanding of the role of inflammation, infection, and mitochondrial dysfunction in the development of cancer cells.

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

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.

Heavy ion beams are a useful tool for conducting high energy density physics (HEDP) experiments. Target heating can be enhanced by beam compression, because a shorter pulse diminishes hydrodynamic expansion during irradiation. A conceptual design is introduced to compress similar to 100 MeV/u to similar to GeV/u heavy ion beams using a wedge. By deflecting the beam with a time-varying field and placing a tailor-made wedge amid its path downstream, each transverse slice passes through matter of different thickness. The resulting energy loss creates a head-to-tail velocity gradient, and the wedge shape can be designed by using stopping power models to give maximum compression at the target. The compression ratio at the target was found to vary linearly with (head-to-tail centroid offset/spot radius) at the wedge. The latter should be approximately 10 to attain tenfold compression. The decline in beam quality due to projectile ionization, energy straggling, fragmentation, and scattering is shown to be acceptable for well-chosen wedge materials. A test experiment is proposed to verify the compression scheme and to study the beam-wedge interaction and its associated beam dynamics, which will facilitate further efforts towards a HEDP facility.

Emittance posts limits on the key requirements of final pulse length and spot size on target in heavy ion fusion drivers. In this paper, we show studies on the effect of nonlinear space charge on longitudinal emittance growth in the drift compression section. We perform simulations, using the 3D PIC code WARP, for a high current beam under conditions of bends and longitudinal compression. The linear growth rate for longitudinal emittance turns out to depend only on the peak line charge density, and is independent of pulse length, velocity tilt, and/or the pipe and beam size. This surprisingly simple result is confirmed by simulations and analytic calculations.

A non-invasive approach for the measurement of the kinetic energy of space-charge dominated ion beams is proposed based on measurements of beam current signals at two different positions. We found that the evolution of space-charge waves, which are generated by weak voltage variations in induction accelerator gaps, can be computed precisely by Fourier analysis of the current signals. The beam energy profile can then be derived from the current signals including the effect of transient space-charge waves. Through analytic studies of the space-charge wave propagation, confirmed by 3-D PIC simulations, we show that the current signals are remarkably sensitive to the weak voltage variations.

We have identified a general final compression section for HIF drivers, the section between accelerator and the target. The beams are given a head to tail velocity tilt at the beginning of the section for longitudinal compression, while going through bends that direct it to the target at specific angle. The aim is to get the beams compressed while maintaining a small centroid off-set after the bends. We used a specific example, 1 MJ driver with 500 MeV Rubidium + 1 ion beams. We studied the effect of minimizing dispersion using different bend strategies, and came up with a beamline point design with adiabatic bends. We also identified some factors that lead to emittance growth as well as the minimum pulse length and spot size on the target.