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Androgens, through their actions on the androgen receptor (AR), are required for the development of the prostate and contribute to the pathologic growth dysregulation observed in prostate cancers. Consequently, androgen ablation has become an essential component of the pharmacotherapy of prostate cancer. In this study, we explored the utility of targeting processes downstream of AR as an alternate approach for therapy. Specifically, we show that the serum and glucocorticoid-regulated kinase 1 (SGK1) gene is an androgen-regulated target gene in cellular models of prostate cancer. Furthermore, functional serum- and glucocorticoid-regulated kinase 1 (SGK1) protein, as determined by the phosphorylation of its target Nedd4-2, was also increased with androgen treatment. Importantly, we determined that RNA interference–mediated knockdown of SGK1 expression attenuates the androgen-mediated growth of the prostate cancer cell line LNCaP. Given these findings, we explored the utility of SGK1 as a therapeutic target in prostate cancer by developing and evaluating a small-molecule inhibitor of this enzyme. From these studies emerged GSK650394, a competitive inhibitor that quantitatively blocks the effect of androgens on LNCaP cell growth. Thus, in addition to androgen ablation, inhibition of pathways downstream of AR is likely to have therapeutic utility in prostate cancer. [Cancer Res 2008;68(18):7475–83]

Supplementary Data from Development of a Small-Molecule Serum- and Glucocorticoid-Regulated Kinase-1 Antagonist and Its Evaluation as a Prostate Cancer Therapeutic

The RAS/MAPK pathway is one of the most commonly dysregulated signaling pathways found in human cancers. KRAS, HRAS and NRAS genes, which play a critical role in transducing the signal from receptor tyrosine kinases to the downstream target proteins such as BRAF and MEK1/2, are frequently mutated in most solid tumors. The extracellular signal-regulated kinases, ERK1 and ERK2 (ERK1/2), which are downstream of MEK and RAF kinases, serve as a key node in this crucial signaling pathway in tumor cells. Targeting ERK1/2 offers a promising therapeutic strategy for a broad range of cancers, particularly the ones driven by RAS mutations, and in overcoming resistance to MEK/BRAF inhibitors. In this report, we present discovery and characterization of a novel orally bioavailable compound, ASN007, that showed potent inhibition of ERK1/2 kinases in a biochemical assay with IC50 = 2 nM for both targets. Interestingly, ASN007 displayed a much slower dissociation rate (longer target residence time) as compared to other ERK inhibitors. ASN007 showed potent antiproliferative activity that was selective for RAS/MAPK pathway dependent cancer cell lines, and did not affect the growth of cell lines harboring PI3K pathway or receptor tyrosine kinase mutations. Of note, ASN007 showed much more potent and broader activity against a panel of RAS mutant cell lines as compared to other ERK1/2 inhibitors. In a panel of 96 KRAS mutant cell lines, ASN007 showed strong activity across all KRAS subtype mutations, including G12C, G12D, G12V and G13D. In tumor xenograft models harboring KRAS and NRAS mutations, a daily oral dosing regimen of ASN007 provided strong tumor growth inhibition and was well tolerated. ASN007 also showed strong single-agent antitumor activity in colorectal PDX models with various KRAS subtype mutations including G12C, G12D, and G12S. A clinical phase 1 trial of ASN007 in patients with BRAF and KRAS, HRAS and NRAS mutant melanoma, colorectal, non-small lung and pancreatic cancers is currently ongoing (NCT03415126). Citation Format: Sanjeeva P. Reddy, Scott K. Thompson, Helen Usansky, Roger A. Smith, Louis Denis, Sandeeep Gupta. ASN007, an oral ERK1/2 inhibitor, shows strong antitumor activity across a panel of KRAS subtype mutant cancer models [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B05.

The RAS/RAF/MEK pathway is frequently dysregulated in a broad range of cancers through mutations in several targets such as KRAS, NRAS, HRAS, BRAF, NF1 and receptor tyrosine kinases. Although MEK and BRAF inhibitors improve the outcome for patients with BRAF mutant melanoma and lung cancers, the duration of response is limited due to reactivation of the pathway. The extracellular-signal-regulated kinases, ERK1 and ERK2 (ERK1/2), which are downstream of MEK and BRAF kinases, serve as a key node in this crucial signaling pathway. Targeting ERK1/2 offers a promising therapeutic strategy for a broad range of cancers, particularly the ones driven by RAS mutations, and in overcoming resistance to MEK/BRAF inhibitors. Here, we present discovery and characterization of a novel orally bioavailable compound, ASN007, that showed inhibition of ERK1/2 kinases in a biochemical assay with IC50 = 2 nM for both targets. Interestingly, ASN007 displayed a much slower dissociation rate (longer target residence time) as compared to other ERK inhibitors. ASN007 inhibited the phosphorylation of ERK1/2 substrates such as RSK1, FRA1 and Elk1 in various cell lines with low nM IC50 values. ASN007 showed potent anti-proliferative activity that was selective for MAPK-pathway dependent cancer cell lines. Indeed, ASN007 showed much more robust activity against RAS mutant cell lines as compared to other ERK1/2 inhibitors. In a daily oral dosing regimen, ASN007 demonstrated strong tumor growth inhibition in BRAF, KRAS and NRAS mutant xenograft models in mice and was well tolerated at efficacious doses. Of note, ASN007 showed strong efficacy using various intermittent dosing regimens. It also showed a strong anti-tumor activity in colorectal PDX models with various KRAS mutations. Furthermore, ASN007 showed regression of tumor growth in a melanoma PDX model that was resistant to both MEK and BRAF inhibitors. In addition to the single agent activity, greater efficacy was observed when ASN007 was combined with immunotherapeutic agents such as an anti-PD-1 antibody or an IDO-1 inhibitor. ASN007 does not exhibit significant inhibition of CYP enzymes or the hERG channel. A Phase 1 trial with ASN007 in patients with BRAF and K-, H- and N-RAS mutant melanoma, colorectal, non-small lung and pancreatic cancers is being planned. Citation Format: Sanjeeva P. Reddy, Niranjan S. Rao, Roger A. Smith, Scott K. Thompson, Sarper Toker. Strong antitumor activity of ASN007, an oral ERK1/2 inhibitor, in PDX tumor models with MAP kinase pathway alterations including KRAS mutations [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 5783.

The extracellular-signal-regulated kinases, ERK1 and ERK2 (ERK1/2), are key players in the RAS/RAF/MEK signaling pathway. This pathway is frequently hyper-activated in a wide range of cancers through mutations in upstream targets such as BRAF, RAS, and receptor tyrosine kinases. Inhibition of ERK1/2 offers a promising therapeutic strategy for these cancers, particularly the ones driven by RAS mutations. In order to inhibit this critical node of the RAS/RAF/MEK pathway, we discovered and characterized ASN007, a novel compound that shows potent inhibition of ERK1/2 kinases in a biochemical assay with low single-digit nM IC50 values. ASN007 showed a slow dissociation rate (long target residence time) as compared to several other known ERK inhibitors. In mechanistic cell-based assays, ASN007 inhibited the phosphorylation of ERK1/2 substrates such as RSK1, FRA1, and Elk1 in various cell lines. ASN007 showed single-digit nanomolar antiproliferative activity that was selective for MAPK-pathway dependent cancer cell lines. Indeed, ASN007 showed much more potent activity against a broader panel of KRAS, NRAS, and HRAS mutant cell lines compared to other ERK1/2 inhibitors such as BVD-523 and GDC-0994. In a daily oral dosing regimen, ASN007 demonstrated strong inhibition of tumor growth in multiple BRAF and KRAS mutant xenograft models in mice and was well tolerated at efficacious doses. Of note, ASN007 also showed strong efficacy using various intermittent dosing regimens. ASN007 has a good pharmacokinetic profile in preclinical species and does not exhibit significant inhibition of CYP enzymes or hERG channel. IND-enabling studies are ongoing in preparation for a phase 1 clinical trial to evaluate oral ASN007 in patients with BRAF and K- , H- , and N-RAS mutant cancers Citation Format: Sanjeeva P. Reddy, Dhanalakshmi Sivanandhan, Purushottam Dewang, Niranjan Rao, Roger A. Smith, Scott K. Thompson. ASN007, a novel oral ERK inhibitor, shows robust antitumor activity in RAS mutant cancer models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B150.

The RAS-RAF-MEK and PI3K-AKT-mTOR pathways are two major signaling pathways involved in human cancer. Components of these two pathways are frequently mutated in a wide variety of solid tumors. Concurrent double mutations in the two pathways are also observed quite often in a broad range of tumor types. Additionally, inhibition of one of these pathways often leads to the upregulation of the other pathway and development of resistance. In preclinical models, combined inhibition of both pathways has been shown to impart greater efficacy as compared to inhibition of either pathway alone. ASN003 is a novel, highly selective, small-molecule inhibitor of both RAS-RAF and PI3K pathways, discovered using a rational design approach. ASN003 shows potent inhibitory activity against B-RAF and PI3K kinases (low nM IC50). Within the PI3K family, ASN003 has high selectivity for inhibition of PI3Kα and PI3Kδ over PI3Kβ. In a panel of 292 kinases, ASN003 showed high selectivity for inhibiting B-RAF and PI3 kinases, and associated mutant kinases. In cell-based mechanistic studies, ASN003 inhibited phosphorylation of ERK, AKT and S6, and showed strong antiproliferative activity (IC50 = 60-300 nM) in cell lines with B-RAF and PI3K pathway mutations as well as in vemurafenib-resistant cell lines. In pharmacodynamic studies in multiple tumor models, ASN003 showed strong inhibition of the phosphorylation of downstream targets of B-RAF and PI3K, confirming appropriate target engagement. In in vivo efficacy studies, ASN003 showed strong tumor growth inhibition or regression in multiple tumor xenograft models, including A375 (B-Raf V600E mutation), RKO (B-Raf V600E and PIK3CA mutations), and A2058 (B-Raf V600E mutation and PTEN loss). We now report that ASN003 also showed strong tumor growth inhibition (>80%) in a patient-derived xenograft (PDX) model established from a relapsed patient with progressive B-Raf mutant melanoma who showed initial response to vemurafenib. Sequencing analysis showed that the vemurafenib resistant tumor acquired a concurrent PIK3CA mutation. Dual targeting of the B-RAF and PI3K pathways with ASN003 has the potential to treat and/or prevent the acquired resistance to selective B-RAF inhibitors, and may also treat a broader patient population and provide greater efficacy and survival benefit than selective B-RAF inhibitors or selective PI3K pathway inhibitors alone. ASN003 is currently in Phase I clinical development in patients with advanced solid tumors, including tumors with B-Raf V600 mutation, PI3 kinase pathway alterations or PTEN loss. Citation Format: Scott K. Thompson, Roger A. Smith, Niranjan Rao, Michael J. Wick, Sanjeeva P. Reddy. ASN003, a highly selective inhibitor of B-Raf and PI3 kinases, shows strong antitumor activity in B-Raf inhibitor resistant patient-derived xenograft models [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 158. doi:10.1158/1538-7445.AM2017-158

Spleen tyrosine kinase (SYK) and Janus kinase (JAK) play important roles in the pathogenesis of various types of lymphomas, solid tumors, and myeloproliferative and inflammation disorders. Inhibition of both SYK and JAK kinases has been shown to suppress tumor growth in various preclinical and/or clinical studies. ASN002 is a novel and potent dual inhibitor of SYK and JAK kinases with IC50 values of 4-46 nM in biochemical assays. In mechanistic cell-based studies, ASN002 strongly suppressed the SYK and JAK family kinase signaling pathways as measured in pLAT and pSTAT assays, respectively. When profiled in a panel of 178 cell lines, the compound showed strong anti-proliferative activity in many lymphoid/leukemia cell lines, including SU-DHL-6, SU-DHL-4, OCI-LY10, H929 and Pfeiffer. The proliferation of many solid tumor cell lines representing bladder, lung and colorectal cancer tumor types was also potently inhibited. Importantly, it also strongly inhibited the growth of ibrutinib and idelalisib resistant cell lines. ASN002 was highly efficacious in inhibiting the tumor growth in several tumor xenograft models which include Pfeiffer (diffuse large B-cell lymphoma, DLBCL), SU-DHL-6 (DLBCL), H929 (myeloma), HEL (erythroleukemia), and RT112 (bladder). ASN002 shows little to no inhibition of CYP450 isozymes suggesting low potential for drug-drug interactions. ASN002 is currently being evaluated in a Phase I/II clinical study in patients with lymphomas (DLBCL, mantle cell lymphoma and follicular lymphoma) and solid tumors. Citation Format: Sanjeeva P. Reddy, Niranjan Rao, David Zammit, Scott K. Thompson, Roger A. Smith, Louis Denis. ASN002: A novel dual SYK/JAK inhibitor with strong antitumor activity in both hematological and solid tumor xenograft models [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 4204. doi:10.1158/1538-7445.AM2017-4204

Various genes in RAS-RAF and PI3K pathways are frequently mutated in a wide variety of solid tumors. Concurrent double mutations are also observed quite often in a broad range of tumor types. Combined inhibition of both pathways has been shown to impart greater efficacy in multiple tumor models in animals. In order to simultaneously block these pathways, we have discovered and characterized several molecules showing dual inhibition of the RAS-RAF and PI3K pathways. ASN003 has been identified as a lead compound with potent and highly selective inhibitory activity against B-RAF, PI3K and mTOR kinases (low nM IC50). Within the PI3K family, ASN003 has high selectivity for inhibition of PI3Kα and PI3Kδ over PI3Kβ. In cell-based mechanistic studies, ASN003 did not increase phospho-ERK levels in the KRAS mutant cell line HCT116 and thus may not cause paradoxical activation of RAS/MAPK pathway in tumors. Consistent with its dual inhibition profile, ASN003 showed strong antiproliferative activity in cell lines with B-RAF and PI3K pathway mutations as well as vemurafenib (B-RAF inhibitor)-resistant cell lines. ASN003 suppressed the phosphorylation of downstream targets of B-RAF, PI3K and mTOR in pharmacodynamic studies in multiple tumor models, indicating appropriate target engagement. In in vivo efficacy studies, ASN003 showed regression in a B-RAFV600E mutant A375 xenograft model and also caused significant tumor growth inhibition in RKO and A2058 tumor models, which harbor mutations in PIK3CA or PTEN genes, respectively, in addition to the B-RAF V600E mutation. Dual targeting of the B-RAF and PI3K pathways with ASN003 has the potential to treat and/or prevent the acquired resistance to selective B-RAF inhibitors, and may also treat a broader patient population and provide greater efficacy and survival benefit than selective B-RAF inhibitors or selective PI3K pathway inhibitors alone. Due to the lack of paradoxical activation, treatment with ASN003 may not cause skin toxicities seen with pure B-RAF inhibitors. ASN003 is currently in preclinical development and is expected to enter Phase I/II clinical trials by early 2016. Citation Format: Scott K. Thompson, Mahaboobi Jaleel, Vijay Kumar Nyavanandi, Murali Ramachandra, Hosahalli Subramanya, Aravind Basavaraju, Vaibhav Sihorkar, Roger A. Smith, Niranjan Rao, Sandeep Gupta, Sanjeeva P. Reddy. ASN003, a unique B-RAF inhibitor with additional selective activity against PI3K and mTOR kinases, shows strong antitumor activity in multiple xenograft models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B100.

Spleen tyrosine kinase (SYK) and Janus kinase (JAK) play important roles in the pathogenesis of various types of lymphomas, solid tumors, myeloproliferative and inflammation disorders. Inhibition of these targets has been shown to suppress tumor growth in various preclinical and/or clinical studies. Most B-cell malignancies are driven by the B-cell receptor (BCR) pathway, and SYK plays a critical role in this pathway by initiating and amplifying the signal from the receptor. JAK kinases (JAK1, JAK2, JAK3 and TYK2), upon stimulation by various growth factors and cytokines, phosphorylate signal transducers and activators of transcription family proteins (STAT1 -5) which activate various downstream target genes. ASN002 is a novel and potent dual inhibitor of SYK and JAK kinases with IC50 values of 5-46 nM in biochemical assays. In mechanistic cell-based studies involving IgE and cytokine stimulations, ASN002 strongly suppressed the SYK and JAK family kinase signaling pathways measured as pLAT and pSTAT levels, respectively. The compound showed anti-proliferative activity in a broad panel of human cancer cell lines including DHL6, DHL4, OCI-LY10, H929, Pfeiffer, HT-1376, and Lovo, suggesting activity in both solid and hematological tumor types. In a multiple myeloma (H929) xenograft model, ASN002 exhibited significant efficacy in inhibiting tumor growth (>95%). It also significantly delayed the onset of hind limb paralysis in the human erythroleukemia (HEL) mouse model. ASN002 has good oral bioavailability, metabolic stability, is not a Pgp substrate, and shows little to no inhibition of CYP450 isozymes. ASN002 showed a favorable safety profile in rat and dog toxicology studies. A Phase I/II clinical study is being planned for the evaluation of ASN002 in lymphomas (diffuse large B cell lymphoma, mantle cell lymphoma, follicular lymphoma) and solid tumors. Citation Format: Sanjeeva Reddy, Nitin K. Damle, Aranapakam M. Venkatesan, Scott K. Thompson, Niranajan Rao, Roger A. Smith, Sandeep Gupta. ASN002: A novel dual SYK/JAK inhibitor with strong antitumor activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 792. doi:10.1158/1538-7445.AM2015-792

The RAS-RAF-MAPK and PI3K pathways are two major signaling pathways involved in the initiation and progression of a broad range of tumors. These two pathways are frequently activated in the majority of solid tumors through mutations in various components of the pathways. Inhibition of one pathway often leads to the activation of the other pathway. Preclinical studies have shown that simultaneous inhibition of these two pathways leads to greater efficacy in a broader range of tumor types. For example, despite the presence of the B-RAFV600E mutation, colorectal cancer tumors do not respond to B-RAF selective inhibitors. In contrast, the combination of B-RAF or MEK inhibitors with PI3K pathway inhibitors shows significant tumor growth inhibition in colorectal cancer xenograft models in mice. Currently, there are several clinical trials ongoing using combinations of inhibitors targeting both pathways. We employed a rational drug design approach to discover and develop a single compound with dual inhibition of both RAS-RAF-MAPK and PI3K pathways. EN3352 was identified as a lead compound with potent inhibitory activity against B-RAF, PI3K and mTOR kinases, with low nanomolar IC50 values. Profiling of EN3352 in a panel of 292 kinases showed that it is highly selective for these three kinases. Within the PI3K family, EN3352 is selective for inhibition of PI3Kα versus PI3Kβ. EN3352 showed broader anti-proliferative activity in tumor cell lines compared to the B-Raf selective inhibitors, vemurafenib and dabrafenib. EN3352 has good oral bioavailability in preclinical species and showed inhibition of phosphorylation of the downstream targets of B-RAF, PI3K and mTOR in various tumor models in mice. EN3352 showed regression in a B-RAFV600E mutant A375 xenograft model and also showed significant tumor growth inhibition in a RKO model, which harbors mutations in both B-RAF and PIK3CA genes. Dual targeting of the B-RAF and PI3K pathways with EN3352 has the potential to treat and/or prevent the acquired resistance to selective B-RAF inhibitors, and may also treat a broader patient population and provide greater efficacy and survival benefit than selective B-RAF inhibitors or selective PI3K pathway inhibitors alone. (Disclosure: Funding for this research was provided by Endo Pharmaceuticals Inc.) Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B274. Citation Format: Sanjeeva P. Reddy, Mahaboobi Jaleel, Vijay K. Nyavanandi, Murali Ramachandra, Hosahalli Subramanya, Aravind Basavaraju, Vaibhav Sihorkar, Roger A. Smith, Sandeep Gupta, Scott K. Thompson. Discovery and characterization of a highly selective inhibitor of B-RAF, PI3K, and mTOR kinases with antitumor activity in B-RAF and B-RAF/PI3K pathway double mutant xenograft models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B274.