Neal Rosen

Synthesis and evaluation of geldanamycin-estradiol hybrids

Geldanamycin (GDM) binds to the Hsp90 chaperone protein and causes the degradation of several important signalling proteins. A series of novel estradiol-geldanamycin hybrids has been synthesized and evaluated for their ability to induce the selective degradation of the estrogen receptor (ER). The hybrid compounds are active and more selective than the parent causing degradation of ER and HER2, but not other GDM targets.

Synthesis and evaluation of geldanamycin–testosterone hybrids

Geldanamycin (GDM) binds to the Hsp90 chaperone protein resulting in the degradation of several important signaling proteins. A series of GDM-testosterone linked hybrids has been synthesized and evaluated for activity against prostate cancer cell lines. The hybrid with the greatest activity exhibits potent and selective cytotoxicity against prostate cancer cells containing the androgen receptor.

PTEN Loss-of-Function Alterations Are Associated With Intrinsic Resistance to BRAF Inhibitors in Metastatic Melanoma

PurposenThe clinical use of BRAF inhibitors in patients with melanoma is limited by intrinsic and acquired resistance. We asked whether next-generation sequencing of pretreatment tumors could identify coaltered genes that predict for intrinsic resistance to BRAF inhibitor therapy in patients with melanoma as a prelude to rational combination strategies.nnnPatients and MethodsnWe analyzed 66 tumors from patients with metastatic BRAF-mutant melanoma collected before treatment with BRAF inhibitors. Tumors were analyzed for > 250 cancer-associated genes using a capture-based next-generation sequencing platform. Antitumor responses were correlated with clinical features and genomic profiles with the goal of identifying a molecular signature predictive of intrinsic resistance to RAF pathway inhibition.nnnResultsnAmong the 66 patients analyzed, 11 received a combination of BRAF and MEK inhibitors for the treatment of melanoma. Among the 55 patients treated with BRAF inhibitor monotherapy, objective responses, as assessed by Response Evaluation Criteria in Solid Tumors (RECIST), were observed in 30 patients (55%), with five (9%) achieving a complete response. We identified a significant association between alterations in PTEN that would be predicted to result in loss of function and reduced progression-free survival, overall survival, and response grade, a metric that combines tumor regression and duration of treatment response. Patients with melanoma who achieved an excellent response grade were more likely to have an elevated BRAF-mutant allele fraction.nnnConclusionnThese results provide a rationale for cotargeting BRAF and the PI3K/AKT pathway in patients with BRAF-mutant melanoma when tumors have concurrent loss-of-function mutations in PTEN. Future studies should explore whether gain of the mutant BRAF allele and/or loss of the wild-type allele is a predictive marker of BRAFi sensitivity.

Abstract 4078: Tumors with class 3 BRAF mutants are sensitive to the inhibition of activated RAS

Approximately two hundred mutant BRAF alleles have been identified in human tumors. Physiologic activation of RAF isoforms requires RAS-dependent induction of their dimerization. Activating BRAF mutants cause ERK dependent feedback inhibition of RAS.GTP and are RAS independent. As we have shown previously, they signal either as active monomers or RAS-independent constitutively active dimers. Here, we characterized a third class of BRAF mutants—those that have impaired kinase activity or are kinase dead. These class 3 BRAF mutants are sensitive to ERK-mediated feedback and they function in a RAS-dependent manner. In tumors, they bind more tightly to active RAS, thus enhancing their heterodimerization with CRAF. This is associated with the amplification of RAS-RAF-MEK-ERK signaling. Since these mutants are sensitive to ERK-dependent feedback inhibition of RAS, their enhancement of ERK signaling in tumors requires concurrent dysregulation of RAS activation. Thus, melanomas with Class 3 mutations usually harbor coexistent RAS mutation or NF1 mutants/deletion, whereas receptor tyrosine kinase signaling is activated in lung and colorectal cancers with these mutants. Our model suggests that these tumors will be sensitive to the inhibition of RAS activation. Currently, no direct inhibitors of RAS activation are available. However, in support of this idea, inhibitors of activated RTK signaling in carcinomas with Class 3 BRAF mutants and wild type RAS is sufficient to markedly inhibit ERK signaling and their growth in in vivo murine models and in patients. We have thus defined a third subset of BRAF mutants, which is RAS-dependent. Tumors harboring such mutants are sensitive to tyrosine kinase inhibitors in tumors expressing wild type RAS and NF1. Citation Format: Zhan Yao, Rona Yaeger, Vanessa S. Rodrik-Outmezguine, Anthony Tao, Neilawattie M. Torres, Matthew T. Chang, Matthias Drosten, Huiyong Zhao, Fabiola Cecchi, Todd Hembrough, Judith Michels, Herve Baumert, Linde Miles, Naomi M. Campbell, Elisa de Stanchina, David B. Solit, Mariano Barbacid, Barry S. Taylor, Neal Rosen. Tumors with class 3 BRAF mutants are sensitive to the inhibition of activated RAS [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 4078. doi:10.1158/1538-7445.AM2017-4078

417 A Mechanistic Logic for Dual Targeting of HER2 and PI3K/AKT/mTOR Signaling in HER2 Amplified Breast Cancer

S. Chandarlapaty, V. Rodrik-Outmezguine, M. Maurizio Scaltriti, R. Sakr, M. Will, D. Giri, C. Hudis, J. Baselga, T. King, N. Rosen. Memorial Sloan-Kettering Cancer Center, Human Oncology and Pathogenesis Program, New York, USA; Memorial Sloan-Kettering Cancer Center, Molecular Pharmacology, New York, USA; Massachusetts General Hospital Cancer Center, Medicine, Boston, USA; Memorial Sloan-Kettering Cancer Center, Surgery, New York, USA; Memorial Sloan-Kettering Cancer Center, Pathology, New York, USA; Memorial Sloan-Kettering Cancer Center, Medicine, New York, USA

Abstract P4-08-02: Reactivation of oncogenic signaling through mTOR inhibitors-induced feedback adaptations.

mTORC1 inhibitors (or rapalogs) relieve feedback inhibition of receptor tyrosine kinases (RTKs) and cause mTORC2-dependent phosphorylation of AKT S473 and activation of AKT kinase and signaling. The relief of feedback inhibition of mTORC2/AKT signaling has been shown to abolish the antitumor activity of rapalogs and has often been proposed as a potential mechanism of resistance to these compounds. mTOR kinase inhibitors that inhibit both mTORC1 and mTORC2 complexes have now been developed to circumvent these problems. mTOR kinase inhibitors block mTORC1 and mTORC2 and thus, do not cause the mTORC2 activation of AKT observed with rapamycin. We now show, however, that these novel classes of drugs have a biphasic effect on AKT. Inhibition of mTORC2 leads to AKT S473 dephosphorylation and a rapid but transient inhibition of AKT T308 phosphorylation and AKT signaling. However, inhibition of mTOR kinase, as observed with rapalogs, also relieves feedback inhibition of RTKs leading to subsequent PI3K activation and rephosphorylation of AKT but only at T308, which is sufficient to reactivate AKT activity and signaling. Thus, catalytic inhibition of mTOR kinase leads to a new steady state characterized by profound inhibition of mTORC1 and accumulation of activated AKT phosphorylated on T308 but not S473. We now confirm that the reactivation of AKT signaling is a mechanism of acquired resistance to rapalogs. We observe an induction of phospho-RTKs in rapamycin-resistant cells leading to both AKT and ERK signaling pathway activation enhancing cell survival. Combined inhibition of mTOR and the induced RTKs can prevent AKT reactivation and fully abolishes AKT signaling. These results reveal the adaptive capabilities of oncogenic signaling networks, highlighting the possible need for combinatorial approaches to block feedback-regulated pathways. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-08-02.

Abstract 1223: Relief of feedback inhibition of RTK and RAS signaling in V600E BRAF melanomas exposed to RAF inhibitors buffers their effects on signaling

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnActivation of ERK signaling causes feedback inhibition of receptor tyrosine kinase and RAS signaling by multiple mechanisms and limits the steady-state output of the pathway. The V600E BRAF mutant does not depend on RAS-dependent dimerization and is not susceptible to feedback inhibition of RAS. In V600E BRAF melanomas, ERK pathway output and the expression of ERK genes such as DUSP6 and members of the SPROUTY family are markedly elevated. In these tumors, RTK signaling and RAS activation are actively suppressed. In the context of elevated DUSP6, RAF inhibitors rapidly inhibit ERK phosphorylation and output. This relieves the feedback inhibition of various RTKs and induces RAS activity and the formation of activated wild type RAF dimers that are insensitive to RAF-inhibitors. This results in a rebound in ERK phosphorylation and output that is sensitive to MEK, but not RAF inhibitors and suggests that the latter cannot maximally inhibit ERK signaling in these tumors. In support of this idea, combined treatment with MEK and RAF inhibitors reduces ERK output and the growth of V600E BRAF melanoma xenografts to a greater degree than either alone.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1223. doi:1538-7445.AM2012-1223

Abstract LB-20: mTOR kinase inhibition relieves feedback suppression of receptor tyrosine kinase leading to a biphasic regulation of AKT signaling in breast cancer cells

Activation of mitogenic signaling by receptor tyrosine kinases (RTKs) is regulated by negative feedback. Feedback is in part mediated by mTORC1/S6K dependent downregulation of IRS proteins which downregulate IGF/insulin receptor signaling. We have found that mTORC1 inhibitors or rapalogs relieve feedback inhibition of receptor tyrosine kinases and cause mTORC2-dependent phosphorylation of AKT S473 leading to activation of AKT kinase and signaling. This relief of feedback inhibition of mTORC2/AKT signaling has been associated with the limited clinical efficacy of these compounds. mTOR kinase inhibitors have now been developed to circumvent these problems; they block mTORC1 and mTORC2 and thus do not cause the mTORC2-dependent activation of AKT observed with the mTORC1 inhibitors. We now show, however, that these drugs have a biphasic effect on AKT. Inhibition of mTORC2 leads to AKT S473 dephosphorylation and a rapid but transient inhibition of AKT T308 phosphorylation and AKT signaling. Inhibition of mTOR kinase also relieves feedback suppression of RTKs leading to subsequent PI3K activation and rephosphorylation of AKT T308 sufficient to reactivate AKT activity and signaling. Thus, catalytic inhibition of mTOR kinase leads to a new steady state characterized by profound inhibition of mTORC1 and accumulation of activated AKT phosphorylated on T308 but not S473. The addition of RTK inhibitors can prevent this reactivation of AKT signaling and cause profound cell death and tumor regressions in vivo. These results highlight the possible need for combinatorial approaches to block feedback-regulated pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-20. doi:1538-7445.AM2012-LB-20

Abstract A165: mTOR kinase inhibition causes feedback-dependent biphasic regulation of AKT signaling.

mTORC1 inhibitors relieve feedback inhibition of receptor tyrosine kinases (RTKs) and cause mTORC2-dependent phosphorylation of AKT S473 and activation of AKT kinase and signaling. mTOR kinase inhibitors block mTORC1 and mTORC2 and thus do not cause the mTORC2 activation of AKT observed with the mTORC1 inhibitor rapamycin. We now show, however, that these drugs have a biphasic effect on AKT. Inhibition of mTORC2 leads to AKT S473 dephosphorylation and a rapid but transient inhibition of AKT T308 phosphorylation and AKT signaling. However, inhibition of mTOR kinase also relieves feedback inhibition of RTKs leading to subsequent PI3K activation and rephosphorylation of AKT T308 sufficient to reactivate AKT activity and signaling. Thus, catalytic inhibition of mTOR kinase leads to a new steady state characterized by profound inhibition of mTORC1 and accumulation of activated AKT phosphorylated on T308 but not S473. Combined inhibition of mTOR kinase and the induced RTKs fully abolishes AKT signaling and results in profound cell death and tumor regression in vivo. These results reveal the adaptive capabilities of oncogenic signaling networks, as AKT signaling may be driven by a loss-of-feedback-induced AKT species lacking S473 phosphorylation. The addition of RTK inhibitors can prevent this reactivation of AKT signaling and cause profound cell death and tumor regressions in vivo, highlighting the possible need for combinatorial approaches to block feedback-regulated pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A165.

Abstract 917: mTOR kinase inhibition relieves feedback inhibition of RTK and PI3K signaling in breast cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnActivation of mitogenic signaling by receptor tyrosine kinases is regulated by negative feedback. Feedback is in part mediated by mTORC1/S6K dependent downregulation of IRS proteins which downregulate IGF/insulin receptor signaling. Rapamycin, an mTORC1 inhibitor, relieves this feedback and causes induction of phosphorylation of AKT at Serine 473 via IGF activation of mTORC2 leading to the activation of AKT kinase and signaling. This relief of feedback inhibition of mTORC2/AKT signaling may attenuate or abolish the antitumor activity of rapamycin. mTOR kinase inhibitors that inhibit both mTORC1 and mTORC2 complexes have now been developed to circumvent these problems. We now find that AZD8055 is a potent and selective inhibitor of both mTORC1 and mTORC2 and thus abolishes AKT phosphorylation at Ser473 as well as 4EBP1 and S6K phosphorylation, in contrast to rapamycin which effectively inhibits only the last and which activates the first. This drug potently inhibited the proliferation of tumor cells, especially those in which PI3K/AKT signaling is dysregulated and sometimes induced their death. However, AZD8055 does potently reactivate receptor tyrosine kinase signaling. It enhanced receptor tyrosine kinase phosphorylation and expression and caused a profound, HER-kinase dependent activation of ERK and PI3K signaling. It reactivated phosphorylation of AKT Thr308 and AKT substrates, but not that of Ser473 AKT. These data suggest that inhibition of the phosphorylation of AKT at Serine 473 is not sufficient to inhibit AKT signaling. Furthermore, relief of feedback inhibition of RTK function by mTOR kinase inhibitors leads to reactivation of both ERK and AKT signaling.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 917. doi:10.1158/1538-7445.AM2011-917