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Osimertinib is an oral, third-generation, irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) that selectively inhibits both EGFR-TKI–sensitizing and EGFR T790M–resistance mutations with lower activity against wild-type EGFR and has demonstrated efficacy in non–small cell lung cancer (NSCLC) CNS metastases. The sensitizing mutations, the in-frame deletions in exon 19 and the L858R point mutation in exon 21, represent between 80% and 90% of all EGFR mutations. The remaining 10% to 20% are referred to as uncommon activating mutations and are a diverse group of mutations in exons 18 to 21 within the kinase domain of the EGFR gene. Excluding those found as insertion mutations in exon 20, the uncommon mutations involving codons G719, S768, and L861 are the most prevalent. Although the efficacy of EGFR-TKIs for the common EGFR mutations is well established, much less is known about rare EGFR mutations, such as exon 20 insertions, G719X, L861Q, S768I, as most of the data consist of single case reports or small case series. Using available patient-derived xenografts (PDX) and cell lines derived from two of these PDXs that harbor the G719X mutation, we have evaluated in vitro and in vivo the preclinical activity of osimertinib. We report osimertinib inhibits signaling pathways and cellular growth in G719X-mutant cell lines in vitro and demonstrate sustained tumor growth inhibition of PDX harboring the G719X mutation alone or in combination with L861Q and S768I. Together, these data support clinical testing of osimertinib in patients with uncommon EGFR NSCLC.

Third-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs), including osimertinib, an irreversible EGFR-TKI, are important treatments for non-small cell lung cancer with EGFR-TKI sensitizing or EGFR T790M resistance mutations. While patients treated with osimertinib show clinical benefit, disease progression and drug resistance are common. Emergence of de novo acquired resistance from a drug tolerant persister (DTP) cell population is one mechanism proposed to explain progression on osimertinib and other targeted cancer therapies. Here we profiled osimertinib DTPs using RNA-seq and ATAC-seq to characterize the features of these cells and performed drug screens to identify therapeutic vulnerabilities. We identified several vulnerabilities in osimertinib DTPs that were common across models, including sensitivity to MEK, AURKB, BRD4, and TEAD inhibition. We linked several of these vulnerabilities to gene regulatory changes, for example, TEAD vulnerability was consistent with evidence of Hippo pathway turning off in osimertinib DTPs. Last, we used genetic approaches using siRNA knockdown or CRISPR knockout to validate AURKB, BRD4, and TEAD as the direct targets responsible for the vulnerabilities observed in the drug screen.

Tyrosine kinase inhibitors (TKI) targeting mutant EGFR in non–small cell lung cancer (NSCLC) have been successful to control cancer growth, but acquired resistance inevitably occurs, including mutations directly on EGFR, for example, T790M and C797S. Strategies to prevent such acquired mutations by reducing mutant-EGFR expression have met limited success. Here, we propose a new model of mutant-EGFR trafficking and demonstrate that clathrin inhibition induces rapid degradation across a large panel of endogenous mutant-EGFR (Ex19del, L858R, and Ex20Ins). This panel included mutant-EGFR (T790M) resistant to the first- and second-generation EGFR inhibitors and to the third-generation TKI osimertinib and occurs through both mutational (C797S) and nonmutational EGFR mechanisms. Clathrin-mediated endocytosis inhibition of mutant EGFR induced a macropinocytosis-dependent lysosomal pathway associated with a loss of mutant-EGFR–dependent signaling (pAKT, pERK). Moreover, induction of this macropinocytic pathway led to robust apoptosis-dependent death across all mutant-EGFR cell lines tested, including those resistant to TKIs. We, therefore, propose a novel strategy to target mutant-EGFR refractory to approved existing TKI treatments in NSCLC and where new treatment strategies remain a key area of unmet need. Significance:These findings extend our mechanistic understanding of NSCLC mutant EGFR trafficking biology, the role that trafficking may play in resistance of mutant EGFR to tyrosine kinase inhibitors, and provide new therapeutic and biological insights to tackle this fundamental issue and improve benefit to patients. Cancer Res; 78(12); 3267–79. ©2018 AACR.

Barasertib (AZD1152), a highly potent and selective aurora kinase B inhibitor, gave promising clinical activity in elderly acute myeloid leukemia (AML) patients. However, clinical utility was limited by the requirement for a 7-day infusion. Here we assessed the potential of a nanoparticle formulation of the selective Aurora kinase B inhibitor AZD2811 (formerly known as AZD1152-hQPA) in preclinical models of AML. When administered to HL-60 tumor xenografts at a single dose between 25 and 98.7 mg/kg, AZD2811 nanoparticle treatment delivered profound inhibition of tumor growth, exceeding the activity of AZD1152. The improved antitumor activity was associated with increased phospho-histone H3 inhibition, polyploidy, and tumor cell apoptosis. Moreover, AZD2811 nanoparticles increased antitumor activity when combined with cytosine arabinoside. By modifying dose of AZD2811 nanoparticle, therapeutic benefit in a range of preclinical models was further optimized. At high-dose, antitumor activity was seen in a range of models including the MOLM-13 disseminated model. At these higher doses, a transient reduction in bone marrow cellularity was observed demonstrating the potential for the formulation to target residual disease in the bone marrow, a key consideration when treating AML. Collectively, these data establish that AZD2811 nanoparticles have activity in preclinical models of AML. Targeting Aurora B kinase with AZD2811 nanoparticles is a novel approach to deliver a cell-cycle inhibitor in AML, and have potential to improve on the clinical activity seen with cell-cycle agents in this disease. Mol Cancer Ther; 16(6); 1031–40. ©2017 AACR.

Barasertib (AZD1152), a pro-drug of the highly potent and selective Aurora B kinase inhibitor AZD2811, showed promising clinical activity in relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients administered as a 4-day infusion. To improve potential therapeutic benefit of Aurora B kinase inhibition, a nanoparticle formulation of AZD2811 has been developed to address limitations of repeated intravenous infusion. One of the challenges with the use of nanoparticles for chronic treatment of tumors is optimizing dose and schedule required to enable repeat administration to sustain tumor growth inhibition. AZD2811 gives potent cell growth inhibition across a range of DLBCL cells lines in vitro. In vivo, repeat administration of the AZD2811 nanoparticle gave antitumor activity at half the dose intensity of AZD1152. Compared with AZD1152, a single dose of AZD2811 nanoparticle gave less reduction in pHH3, but increased apoptosis and reduction of cells in G1 and G2–M, albeit at later time points, suggesting that duration and depth of target inhibition influence the nature of the tumor cell response to drug. Further exploration of the influence of dose and schedule on efficacy revealed that AZD2811 nanoparticle can be used flexibly with repeat administration of 25 mg/kg administered up to 7 days apart being sufficient to maintain equivalent tumor control. Timing of repeat administration could be varied with 50 mg/kg every 2 weeks controlling tumor control as effectively as 25 mg/kg every week. AZD2811 nanoparticle can be administered with very different doses and schedules to inhibit DLBCL tumor growth, although maximal tumor growth inhibition was achieved with the highest dose intensities.

EGFR exon 20 insertions (Ex20Ins) account for 4% to 10% of EGFR activating mutations in non–small cell lung cancer (NSCLC). EGFR Ex20Ins tumors are generally unresponsive to first- and second-generation EGFR inhibitors, and current standard of care for NSCLC patients with EGFR Ex20Ins is conventional cytotoxic chemotherapy. Therefore, the development of an EGFR TKI that can more effectively target NSCLC with EGFR Ex20Ins mutations represents a major advance for this patient subset. Osimertinib is a third-generation EGFR TKI approved for the treatment of advanced NSCLC harboring EGFR T790M; however, the activity of osimertinib in EGFR Ex20Ins NSCLC has yet to be fully assessed. Using CRISPR-Cas 9 engineered cell lines carrying the most prevalent Ex20Ins mutations, namely Ex20Ins D770_N771InsSVD (22%) or Ex20Ins V769_D770InsASV (17%), and a series of patient-derived xenografts, we have characterized osimertinib and AZ5104 (a circulating metabolite of osimertinib) activities against NSCLC harboring Ex20Ins. We report that osimertinib and AZ5104 inhibit signaling pathways and cellular growth in Ex20Ins mutant cell lines in vitro and demonstrate sustained tumor growth inhibition of EGFR-mutant tumor xenograft harboring the most prevalent Ex20Ins in vivo. The antitumor activity of osimertinib and AZ5104 in NSCLC harboring EGFR Ex20Ins is further described herein using a series of patient-derived xenograft models. Together these data support clinical testing of osimertinib in patients with EGFR Ex20Ins NSCLC. Mol Cancer Ther; 17(5); 885–96. ©2018 AACR.

Exon 20 insertions (Ex20Ins) have been identified in approximately 5% of epidermal growth factor receptor (EGFR)-mutated lung tumours in patients presenting with non-small cell lung cancer (NSCLC). Several small molecule tyrosine kinase inhibitors (TKIs) have been reported to have pre-clinical activity against such insertions including afatinib, poziotinib, osimertinib, nazartinib, AP32788/TAK-788 and TAS6417. However, there remains a lack of approved treatments for patients with Ex20Ins with early approved EGFR agents appearing to be ineffective in this setting. Poziotinib, osimertinib and AP32788/TAK-788 are undergoing clinical evaluation in patients whose tumours carry Ex20Ins and in some cases clinical responses have been reported giving hope that such insertions can be targeted by small molecules. There is however a need for comparable data across such compounds that would enable understanding of the relative activity of these compounds between Ex20Ins and the wild-type form of EGFR. As many of the Exon 20 insertions are not part of the ATP binding pocket achieving selectivity over wild type EGFR is highly challenging and may limit the clinical utility of agents due to dose limiting EGFR wild-type driven toxicity. A selection of TKIs were profiled for Ex20Ins and wild-type EGFR activity using biochemical, in vitro cellular phosphorylation and proliferation assays. This has enabled us to differentiate the Ex20Ins versus wild-type EGFR selectivity profiles of a range of pre-clinical, clinical and proprietary compounds. As part of this evaluation we utilized a CRISPR CAS9 approach in H2073 EGFR wild-type NSCLC cell line, where we have established cellular disease models against the most prevalent insertions including D770-N771insSVD (22%). Finally, we will show anti-tumour efficacy data for a selection of these inhibitors along with a potential combination approach of osimertinib and cetuximab. EGFR D770-N771InsSVD cell phospho IC50 (µM)EGFR WT (H2073) cell phospho IC50 (µM)Fold-EGFR WT margin (cell)afatinib0.0060.00330.5osimertinib0.0940.414.4poziotinib0.00340.00351TAS64170.0230.0672.9AZ62810.0972.223 Citation Format: Richard A. Ward, Ambra Bianco, Nicola Colclough, Darren Cross, Emanuela M. Cuomo, M. Raymond V. Finlay, Martina Fitzek, Nicolas Floc’h, Sladjana Gagrica, Beverley Hammond, Matthew J. Martin, Darren McKerrecher, Daniel J. O’Neill, Jonathan P. Orme, Paul D. Smith, Anna D. Staniszewska, Jelena Urosevic, Nicky Whalley, James W. Yates. Comparative activity profiling of tyrosine kinase inhibitors (TKIs) against exon 20 insertions and the wild-type form of epidermal growth factor receptor (EGFR) [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 4813.

Two first-generation (erlotinib & gefitinib), two second generation (afatinib & dacomitinib) and a third generation (osimertinib) EGFR-TKIs are currently available for the management of EGFR mutation-positive NSCLC. All these TKIs are effective in patients with NSCLC whose tumors harbor the in-frame deletions in exon 19 and the L858R point mutation in exon 21. These two mutations represent 90% of all EGFR mutations. Osimertinib, when used in the front-line setting, has shown more favorable treatment outcomes than first-generation EGFR-TKIs. In approximately 50% of patients, resistance to first and second generation TKI is mediated by the acquisition of the ‘gatekeeper’ mutation T790M. Currently, osimertinib is the only registered EGFR TKI that is active against exon 19 deletions and L858R mutation, regardless of the presence of T790M mutation. While the efficacy of EGFR TKIs for the common EGFR mutations is well established, much less is known about rare EGFR mutations such as exon 20 insertions, G719X, L861Q, S768I, as most of the data consist of single case reports or small case series. This work describes the therapeutic potential of osimertinib in tumors harboring the G719X mutation alone or in combination with L861Q and S768I. Using available patient-derived xenografts (PDX) and cell lines derived from two of these PDX which habor the G719X mutation, we have evaluated in vitro and in vivo the pre-clinical activity of osimertinib. We show that osimertinib inhibits signalling pathways and cellular growth of G719X cell lines in vitro. This translates into sustained tumor growth inhibition in vivo in 3 out of 4 PDX explored (87%, p The work presented herein demonstrates that osimertinib has the potential to improve upon the current treatment options for NSCLC patients whose tumors harbor a G719X mutation, and warrants further clinical investigation. Citation Format: Nicolas Floch, Sue Bickerton, Matthew J. Martin, Darren A. Cross, Paul D. Smith. Osimertinib, an irreversible next generation EGFR tyrosine kinase inhibitor, exerts anti-tumor activity in various preclinical NSCLC models harboring G719X mutant-EGFR [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 1330.

Current treatments for castration-resistant prostate cancer (CRPC) that target androgen receptor (AR) signaling improve patient survival, yet ultimately fail. Here, we provide novel insights into treatment response for the antiandrogen abiraterone by analyses of a genetically engineered mouse (GEM) model with combined inactivation of Trp53 and Pten, which are frequently comutated in human CRPC. These NPp53 mice fail to respond to abiraterone and display accelerated progression to tumors resembling treatment-related CRPC with neuroendocrine differentiation (CRPC-NE) in humans. Cross-species computational analyses identify master regulators of adverse response that are conserved with human CRPC-NE, including the neural differentiation factor SOX11, which promotes neuroendocrine differentiation in cells derived from NPp53 tumors. Furthermore, abiraterone-treated NPp53 prostate tumors contain regions of focal and/or overt neuroendocrine differentiation, distinguished by their proliferative potential. Notably, lineage tracing in vivo provides definitive and quantitative evidence that focal and overt neuroendocrine regions arise by transdifferentiation of luminal adenocarcinoma cells. These findings underscore principal roles for TP53 and PTEN inactivation in abiraterone resistance and progression from adenocarcinoma to CRPC-NE by transdifferentiation. Significance: Understanding adverse treatment response and identifying patients likely to fail treatment represent fundamental clinical challenges. By integrating analyses of GEM models and human clinical data, we provide direct genetic evidence for transdifferentiation as a mechanism of drug resistance as well as for stratifying patients for treatment with antiandrogens. Cancer Discov; 7(7); 736–49. ©2017 AACR. See related commentary by Sinha and Nelson, p. 673. This article is highlighted in the In This Issue feature, p. 653

Up to 20% of non small-cell lung cancers are driven by activating mutations in EGFR (EGFRm). While EGFR tyrosine kinase inhibitors (TKIs) have proven highly effective in the clinic (e.g. gefitinib in first line EGFRm and osimertinib in T790M post-TKI resistance), acquired resistance mechanisms present an obstacle to curing EGFR-driven disease. To-date, approximately 20% of osimertinib-resistant cases in the 2nd line setting are EGFRm/T790M/C797S, and there is currently no approved therapy for these “triple-mutant” (TM) patients. We have obsrerved that the ALK inhibitor brigatinib shows moderate, equipotent activity against both EGFR-TM and the single activating mutant. We used this information to screen a panel of compounds to identify brigatinib-based combinations that could effectively target EGFR-TM-expressing cells. Among the most potent combination partners were selumetinib and other inhibitors of the MAP kinase pathway. These agents were able to synergistically impair growth of TM cells when combined with brigatinib in vitro, and accordingly show prolonged suppression of downstream pathway biomarkers in combination- vs. brigatinib monotherapy-treated cells. Further, we have shown effective combinations can dramatically enhance apoptotic cell death compared to brigatinib treatment alone. Critically, neither monotherapy treatment with effective combination partners, nor combinations based on osimertinib showed growth inhibition in the EGFR triple-mutant setting. Taken together, these data suggest novel therapeutic strategies for patients in this critical area of unmet need that warrant further preclinical testing. Citation Format: Matthew J. Martin, Nicolas Floc'h, Chrysiis Michaloglou, M Raymond Finlay, Richard A. Ward, Paul D. Smith, Darren A. Cross. A screen to target EGFR (C797S) in osimertinib-resistant lung cancer identifies brigatinib-based combinations that synergistically inhibit cell 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 916.

EGFR exon 20 insertion (Ex20Ins) mutations represent a combination of in-frame insertions and/or duplications that account for 4-10% of all EGFR mutants in non-small cell lung cancer (NSCLC). To date, more than one hundred different Ex20Ins mutations have been identified. With the notable exception of the rare A763_Y764insFQEA insertion ( Using CRISPR-Cas 9 genome editing technology, we engineered cellular disease relevant models carrying the most prevalent Ex20Ins mutations, namely Ex20Ins D770_N771InsSVD (22%) or Ex20Ins V769_D770InsASV (17%). Using these models, we show that osimertinib and AZ5104 inhibit signalling pathways and cellular growth of Ex20Ins CRISPR-Cas9 engineered cell lines in vitro. This translates into sustained tumor growth inhibition in vivo in both the Ex20Ins SVD (65%, p EGFR signal. Two patients from the AURA Phase 2 osimertinib trials with plasma positive Ex20Ins (concurrent with Ex19del and T790M) had durable PFS responses of 6.4 and 13.9 months, supporting the premise that osimertinib has the potential to be clinically beneficial in tumors harboring Ex20Ins. The work presented herein demonstrates that osimertinib has the potential to improve upon the current treatment options for NSCLC patients whose tumors harbour an Ex20Ins mutation, and warrants its further clinical investigation. Citation Format: Nicolas Floc'h, Susan Ashton, Ambra Bianco, Nicola Colclough, Darren AE Cross, Maria Emanuela Cuomo, M. Raymond V. Finlay, Matthew J Martin, Ludovic Menard, Darren McKerrecher, Daniel J O'Neill, Jonathan P Orme, Anna D Staniszewska, Richard A Ward, James W T Yates. Osimertinib, an irreversible mutant selective EGFR tyrosine kinase inhibitor, exerts anti tumor activity in NSCLC harbouring exon 20 insertion mutant-EGFR [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 2079. doi:10.1158/1538-7445.AM2017-2079

9030 Background: EGFR exon 20 insertions (Ex20Ins), the 3rd most common EGFR activating mutation, are generally unresponsive to 1st and 2nd generation EGFR-TKIs. Development of EGFR-TKIs that effectively target NSCLC with Ex20Ins mutations represents a major unmet need. Osimertinib is an EGFR TKI approved for the treatment of advanced NSCLC harboring EGFR T790M, but the potential of osimertinib remains to be fully assessed in patients (pts) with Ex20Ins NSCLC. Methods: CRISPR engineered Ex20Ins cell line xenografts representing the two most common Ex20Ins (D770_N771InsSVD and V769_D770InsASV) and pt derived xenograft (PDX) of 3 EGFR Ex20Ins (V769_D770InsASV, M766_A767insASV, H773_V774insNPH) were used for in vivo experiments. Xenografts were treated by oral gavage with vehicle, erlotinib (50 mg/kg/day) or afatinib (20 mg/kg/day), osimertinib metabolite AZ5104 (50 mg/kg/day) and osimertinib (25 mg/kg/day) and assessed for tumor growth inhibition (TGI). Immunoblotting was performed for EGFR and relevant signaling pathways. A pt from whom the V769_D770InsASV Ex20ins PDX was derived was treated on a UC Davis IRB approved protocol with osimertinib at 160 mg PO once-daily (QD). Results: At completion of treatment, QD administration of osimertinib or AZ5104 induced significant TGI in xenografts across the 4 EGFR Ex20ins tested (range 60-95% TGI, p < 0.001 compared to control for all models) that was superior to either afatinib or erlotinib. Robust decrease in p-EGFR, p-ERK, p-Akt, p-Stat3 was observed with osimertinib treatment. The patient corresponding to the V769_D770InsASV Ex20ins PDX treated with osimertinib exhibited clinical improvement and tumor shrinkage; unfortunately he was found to have interstitial pneumonitis that necessitated drug discontinuation. Conclusions: Osimertinib at clinically representative doses has in vivo activity across multiple EGFR Ex20ins that comprising the most common Ex20ins detected in patients (~50% prevalence); metabolite AZ5104 may contribute to efficacy. Tumor shrinkage was observed in a patient with lung cancer harboring an Ex20ins treated for a limited time with osimertinib. Based on this in vivo xenograft and pt data, osimertinib warrants further study in pts with EGFR Ex20ins NSCLC.

International audience; Expression of developmental genes Twist1 and Twist2 is reactivated in many human tumors. Among their oncogenic activities, induction of epithelial to mesenchymal transition is believed to increase cell motility and invasiveness and may be related to acquisition of cancer stem cell phenotype. In addition, Twist proteins promote malignant conversion by overriding two oncogene-induced failsafe programs: senescence and apoptosis. Reactive oxygen species (ROS) are also important mediators of apoptosis, senescence and motility and are tightly linked to disease, notably to cancer. We report here that Twist factors and ROS are functionally linked. In wild type cells both Twist1 and Twist2 exhibit antioxidant properties. We show that Twist-driven modulation of oncogene-induced apoptosis is linked to its effects on oxidative stress. Finally, we identify several targets that mediate Twist antioxidant activity. These findings unveil a new function of Twist factors that could be important in explaining their pleiotropic role during carcinogenesis.

Both the PI3K → Akt → mTOR and mitogen-activated protein kinase (MAPK) signaling pathways are often deregulated in prostate tumors with poor prognosis. Here we describe a new genetically engineered mouse model of prostate cancer in which PI3K-Akt-mTOR signaling is activated by inducible disruption of PTEN, and extracellular signal-regulated kinase 1/2 (ERK1/2) MAPK signaling is activated by inducible expression of a BRAFV600E oncogene. These tissue-specific compound mutant mice develop lethal prostate tumors that are inherently resistant to castration. These tumors bypass cellular senescence and disseminate to lymph nodes, bone marrow, and lungs where they form overt metastases in approximately 30% of the cases. Activation of PI3K → Akt → mTOR and MAPK signaling pathways in these prostate tumors cooperate to upregulate c-Myc. Accordingly, therapeutic treatments with rapamycin and PD0325901 to target these pathways, respectively, attenuate c-Myc levels and reduce tumor and metastatic burden. Together, our findings suggest a generalized therapeutic approach to target c-Myc activation in prostate cancer by combinatorial targeting of the PI3K → Akt → mTOR and ERK1/2 MAPK signaling pathways. Cancer Res; 72(18); 4765–76. ©2012 AACR.

Although the prognosis for clinically localized prostate cancer is now favorable, there are still no curative treatments for castration-resistant prostate cancer (CRPC) and, therefore, it remains fatal. In this study, we investigate a new therapeutic approach for treatment of CRPC, which involves dual targeting of a major signaling pathway that is frequently deregulated in the disease. We found that dual targeting of the Akt and mTOR signaling pathways with their respective inhibitors, MK-2206 and ridaforolimus (MK-8669), is highly effective for inhibiting CRPC in preclinical studies in vivo using a refined genetically engineered mouse model of the disease. The efficacy of the combination treatment contrasts with their limited efficacy as single agents, since delivery of MK-2206 or MK-8669 individually had a modest impact in vivo on the overall tumor phenotype. In human prostate cancer cell lines, although not in the mouse model, the synergistic actions of MK-2206 and ridaforolimus (MK-8669) are due in part to limiting the mTORC2 feedback activation of Akt. Moreover, the effects of these drugs are mediated by inhibition of cellular proliferation via the retinoblastoma (Rb) pathway. Our findings suggest that dual targeting of the Akt and mTOR signaling pathways using MK-2206 and ridaforolimus (MK-8669) may be effective for treatment of CRPC, particularly for patients with deregulated Rb pathway activity. Cancer Res; 72(17); 4483–93. ©2012 AACR.

According to the American Cancer Society approximately 242,000 new cases of prostate cancer will be reported in 2012, making it one of the most frequently diagnosed cancers in men. Moreover, with 1 in 9 men diagnosed with prostate cancer anticipated to die of the disease, prostate cancer is also the second-leading cause of cancer death in men. Although several new treatment options for men with advanced prostate cancer have recently become available, none are as yet curative. Because of the necessity of androgen receptor signaling for all aspects of normal prostate growth, as well as for prostate tumorigenesis, androgen deprivation therapy (i.e., pharmacological depletion of androgens) has long been a mainstay for treatment of men with advanced prostate cancer. However, while androgen deprivation initially leads to tumor regression, ultimately it leads to a highly aggressive form of the disease, which has been called “castration-resistant prostate cancer” (CRPC) to indicate its continued dependence on androgen receptor signaling despite the absence of androgens [1]. The standard of care for CRPC is chemotherapy, which provides a modest improvement in survival but is not curative [2, 3]. In the past few years, several new treatment options for CRPC have received FDA approval, including an immunotherapy agent, namely sipuleucel-T (PROVENGE®), and two agents that inhibit androgen receptor signaling or activity, namely Abiraterone Acetate (ZytigaTM) and enzalutamide (XTANDI®) [4-6]. Although each of these has a significant survival benefit, none are curative for CRPC. Therefore an important challenge is to identify new therapeutic approaches that are curative for CRPC. As an alternative to inhibiting androgen receptor signaling, agents that target critical survival and proliferative pathways that are deregulated in prostate cancer are also being investigated. Among the molecular pathways that are critical for prostate tumorigenesis, loss of function of the PTEN tumor suppressor through its deletion, mutation, or deregulated expression occurs at high frequency [7], leading to aberrant activation of the Akt kinase as well as it downstream effector, the mTOR complex (Figure ​(Figure1).1). Notably, the Akt/mTOR signaling pathway is frequently deregulated in advanced prostate cancer and has been implicated in potentiating androgen receptor signaling; however, targeting this pathway with single agents has proven not to be effective in prostate cancer, as well as is the case for many other cancers [8]. Figure 1 Cross talk between Akt/mTOR and MEK signaling pathways Consequently, in our recent study we asked whether combined inhibition of upstream and downstream nodes in the Akt/mTOR signaling pathway is effective for suppressing CRPC [9]. We used both in vivo studies of a genetically-engineered mouse (GEM) model of CRPC and in vitro studies using human prostate cancer cell lines to investigate the consequences of dual targeting Akt/mTOR signaling, with an inhibitor of Akt, namely, MK-2206, and one that targets mTORC1, namely ridaforolimus (MK-8669) (Figure ​(Figure1).1). We found that dual inhibition of Akt and mTORC1 had very promising therapeutic outcomes both in the preclinical studies in vivo in the GEM models and in the human prostate cancer cell lines in vitro. Why would dual targeting of two kinases in the same pathway yield improved results compared with targeting the pathways individually? In part, this reflects the fact that inhibition of mTORC1 alone has limited efficacy, in part due to feedback activation of PI3K/Akt signaling (Figure ​(Figure1),1), particularly in human prostate cancer cells [9]. On the other hand, inhibition of Akt alone was inefficient for inhibition of mTOR, likely due to MAPK signaling-dependent activation of mTOR (Figure ​(Figure1).1). Consequently, by combining two distinct inhibitors (“vertical inhibition”) we observed an effective neutralization of the Akt/mTOR pathway, which was associated with reduced cell proliferation and inhibition of CRPC tumor growth. How might these findings be applied in the clinic? Currently, several studies have described a crosstalk between the PI3K/Akt/mTOR and AR signaling pathways. In particular, it has been suggested that castration-resistant tumors compensate for reduced AR signaling by activation of Akt/mTOR signaling, which highlights the therapeutic potential of combining inhibition of Akt/mTOR pathways with androgen receptor pathway inhibition. Indeed, Carver et al have suggested that inhibition of the PI3K pathway restores AR signaling in PTEN-deficient prostate cells, and that feedback regulation between AR and PI3K pathways may be reciprocal [10]. Thus, based on recent findings by us and others, we propose that a therapeutic approach using dual inhibitors of Akt/mTOR signaling in combination with inhibitors of androgen receptor signaling, such as Abiraterone Acetate or Enzalutamide, may effectively block androgen signaling while simultaneously inhibiting a Akt/mTOR signaling and therefore an effective treatment course for CRPC. We await the results of the relevant clinical trials.

While most men diagnosed with localized prostate cancer now have favorable outcomes, those with advanced disease often undergo androgen deprivation therapy, which initially leads to tumor regression but ultimately results in the emergence of a castration-resistant form of the disease that is highly metastatic and ultimately fatal. Our research is focused on generating mouse models of advanced prostate cancer to evaluate new therapeutic approaches for its treatment. Toward this end, we have generated state-of the-art genetically-engineered mouse (GEM) models, which are based on biologically-relevant signaling pathways and recapitulate prostate cancer from its earliest to most advanced stages. Indeed these mice recapitulate lethal prostate cancer as they display distant metastases with 100% penetrance as well as disseminated cells in the bone marrow. By pursuing preclinical studies in these GEM mice we have found that combinational targeted of key signaling pathways is significantly more effective than standard of care (i.e., chemotherapy) for blocking cancer progression, improving survival and inhibiting metastases in vivo. However, one of the main obstacles for effectively translating results from preclinical studies in genetically-engineered mouse models is difficulties in overcoming inherent species differences that may preclude direct comparisons from mice to man. To overcome this limitation, we have assembled regulatory networks (called interactomes) for both mouse and human prostate cancer to enable cross-species analyses for identification of master regulators (driver genes) that underlie the therapeutic response. In particular, using these computational resources we have interrogated gene expression profiles from preclinical studies in our genetically-engineered mice, and have identified molecular regulators that predict therapeutic response in the mice and are conserved in human prostate cancer. Our approach highlights the value of cross-species analyses of preclinical studies in mice and human clinical data for predicting drug response. Citation Format: Antonina Mitrofanova, Nicolas Floch, Alvaro Aytes, Michael Shen, Cory Abate-Shen, Andrea Califano. Interrogating therapeutic response for advanced prostate cancer in vivo using cross-species regulatory models. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A29.