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Background: Hyperactivation of the PI3K/AKT pathway correlates with impaired antitumour response, including reduced T cell infiltration into tumour and reduced efficacy of immune checkpoint inhibitors (ICIs). PTEN loss of function, often observed in GBM, may contribute to refractoriness of ICIs in this disease. Methods: The Ice-CAP A2 cohort assessed safety, pharmacodynamic, and preliminary clinical activity of Ipa (200mg or 400mg OD) + A (1200mg Q3W) in pts with potentially resectable relapsed WHO Grade IV GBM. Key inclusion criteria were stable neurological symptoms ≥5 days prior to enrolment, steroid requirement 12wks, both on 400mg Ipa. A 58-year-old man with PTEN loss had MRI at Cycle 5 showing worsening enhancement suggestive of disease progression. Resection of the lesion showed intense lymphocyte infiltration and pathological CR. He is currently on Cycle 18 with no evidence of disease. Conclusion: Combination Ipa+A appears safe and tolerable in GBM pts, with 400mg Ipa OD + 1200mg A Q3W declared as RP2D. PTEN loss may be a promising predictive biomarker for response to combination. An expansion cohort enriched with pts with PTEN loss is ongoing. Cytokine and FACS data will be presented at AACR Table 1.Clinical Benefit Rate of glioblastoma patients stratified according to PTEN aberrationsPTEN statusnBest responseClinical Benefit RatePTEN loss on IHC (H12 wks, ongoingb3 PDPTEN aberration on NGS but PTEN protein expression pending11 PDcPTEN loss of heterozygozity on PCR11 PDWild type PTEN on NGS or IHC (H≥30)33 PDLegend: IHC = immunohistochemistry; NGS = next generation sequencing; PCR = polymerase chain reaction;pCR = pathologic complete response; SD = stable disease; PD = progressive diseaseaExceptional responder with PTEN H=5 on IHC and splice site 75_79+2delGACCTGT on NGSb PTENY68*; c PTENQ298* Citation Format: Crescens Tiu, Andrea Biondo, Liam C. Welsh, Timothy L. Jones, Anna Zachariou, Toby Prout, Alison J. Turner, Robert Daly, Igor Vivanco, Christina Yap, Ben Jenkins, Mateus Crespo, Ruth Riisnaes, Suzanne Carreira, Bora Gurel, Nina Tunariu, Anna Minchom, Udai Banerji, Johann S. de Bono, Juanita S. Lopez. Results of the glioblastoma multiforme (GBM) cohort of phase 1 trial Ice-CAP (NCT03673787): Preliminary evidence of antitumour activity of Ipatasertib (Ipa) and Atezolizumab (A) in patients (pts) with PTEN loss [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT120.

Background: Hyperactivation of the PI3K/AKT pathway correlates with impaired anti-tumor responses, including reduced T cell infiltration into tumor, and reduced efficacy of immune checkpoint inhibitors. Blockade of this pathway synergizes with PD-L1/PD-1 axis blockade preclinically. Methods: This Phase I clinical trial (NCT03673787) assessed the safety, pharmacodynamic, and preliminary clinical activity of Ipa (200mg or 400mg OD) given in combination with A 1200mg q3 wk in refractory pts. Serial paired blood and tumor samples were analysed to interrogate the effect of Ipa on the tumor micro-environment and host immune system prior to the addition of the immune check point inhibitor, A. Results: 18 adult pts were treated in dose escalation. Median age 49 yrs. All pts had ECOG PS 0-1 and median 7 prior therapies. Most common TRAEs (>15%) were mild Gr1-2 diarrhea (56%), rash (50%), fatigue (33%), nausea (33%), raised ALT/AST (33%), headache (28%) and arthralgia (22%). 1 pt had G2 systemic immune activation; 2 pts had G3 rash, both rapidly reversible. 1 DLT of G3 raised ALT seen at 200mg (1 DLT/9 evaluable pts) but none at 400mg (0 DLT/6). Of 14 RECIST evaluable patients, there were 2 confirmed PRs, and 5 SD (clinical benefit rate 50%). Reductions of CD4+FOXP3+ Tregs in tumor microenvironment were seen after 2wks of single agent Ipa, regardless of PIK3/AKT somatic mutation status (Table 1). Responding pts had a >400% median increase in intra-tumoral CD8+ Teff cell infiltration, effectively switching from a desert phenotype to an inflamed phenotype. Paired changes in FACS, transcriptome and cytokine will also be presented.Conclusions: The RP2D of Ipa 400mg OD combination with A was well tolerated with early efficacy signals. Further biomarker work is ongoing and will be evaluated in expansion cohorts. Table 1:Changes in immune cell populations as assessed by multicolour Immunofluorescence in paired biopsies of breast/gynae patients, % change in cell number/mm2 from baseline (median [min,max$])&Post 2 weeks single agent Ipatasertib(n=9)Post 1 cycle of combination Ipatasertib and Atezolizumab(n=7)CD4+FOXP3+Tregs cellsCD 8+ Teff cellsCD4+FOXP3+Tregs cellsCD 8+ Teff cellsIntra-tumourstromaIntra-tumourstromaIntra-tumourstromaIntra-tumourstromaAll patients-23.9*[-89.7, BL0]-30.0*[-91.6, BL0]-37.7*[-84.4, -24.5]-28.4[-92.4, 259.8]335.9[-44.0,BL0]45.4[-51.0, BL0]59.6[-60.6,493.3]64.7[-51.7,293.3]Stratified by somatic PI3K/AKT/PTEN mutational statusPathogenic mutations (mt)11.1[-82.2, BL0]#-10.7[-91.6, BL0]Φnsnsnsns-30.5[-60.6,-0.5]11.3[-51.7,50.0]Wildtype (wt)-63.1[-89.7,19.0]#-47.5[-77.0,11.1]Φnsnsnsns426.5[59.6,493.3]126.7[79.4,293.3]Stratified by responseResponders (PR + SD>4 cycles). 1 ER+ HER2+ breast cancer (wt), 1 ER+ HER2- breast cancer (wt)459.9[426.5,493.3]@103.1[79.4,126.7]Non-responders (PD at 4 cycles) 1 cervical cancer, 4 ER+ breast cancer-0.5[-60.6, 59.6]@30.6[-51.7,293.3]*significant change (p≤0.05; Wilcoxon sign-rank test) from baseline, $maximum values denoted by BL0indicate that the baseline value was zero, and so percentage change from baseline is not defined. For the analysis, the baseline value has been replaced by a nominal value of 0.1 so that a large percentage increase is associated with these cases. Note that these large percentage increases do not affect the non-parametric statistical tests used.#no significant difference in distribution of reduction in intra-tumoural CD4+ FOXP3+Tregsbetween pts with pathogenic mutations in PI3K/AKT and those without (p=0.30; Wilcoxon rank-sum test)Φno significant difference in distribution of reduction in stromal CD4+FOXP3+Tregsbetween pts with pathogenic mutations in PI3K/AKT and those without (p=0.44; Wilcoxon rank-sum test) @ difference between responders and non-responders p=0.083; Wilcoxon rank-sum test)mt pathogenic mutations in PI3K/AKT and PTEN as per COSMIC database present in tumour or PTEN loss by IHC. wt no pathogenic mutations in PI3K/AKT and PTEN as per COSMIC database detected in tumour and intact PTEN expression by IHC. &exploratory analyses with no adjustment for multiple testing Citation Format: Juanita S. Lopez, Andrea Biondo, Crescens Tiu, Mariana Scaranti, Malaka Ameratunga, Anna Zachariou, Alison Turner, Nina Tunariu, Toby Prout, Mona Parmar, Hannah Badham, Karen Swales, Wei Yuan, Ricardo Morilla, Mateus Crespo, Rob Daly, Ines Figueiredo, Bora Gurel, Rita Pereira, Ruth Riisnaes, Igor Vivanco, Anna Minchom, Ben Jenkins, Christina Yap, Udai Banerji, Johann De Bono. Proof-of-concept evidence of immune modulation by blockade of the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway in the phase I dose escalation study of Ipatasertib (Ipa) in combination with atezolizumab (A) in patients (pts) with advanced solid tumors (Ice-CAP) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT140.

Amplification and overexpression of the epidermal growth factor receptor (EGFR) are common in glioblastoma (GBM) and frequently associated with silencing of the phosphatase and tensin homologue (PTEN) tumor suppressor. PTEN silencing has been associated with clinical resistance to EGFR tyrosine kinase inhibitors, in part by raising EGFR levels. Here, we investigated the effect of PTEN on the EGFR signaling complex by EGFR affinity immunopurification and mass spectrometry with and without PTEN knockdown. We identified Ran binding protein 6 (RanBP6), a 125-kDa protein of previously unknown functions, as EGFR interacting protein in PTEN expressing, but not PTEN knockdown cells. Further studies of the effect of RanBP6 on EGFR revealed that RanBP6 depletion by shRNA or CRISPR/Cas9-mediated gene silencing resulted in increased EGFR mRNA levels and upregulation of EGFR promoter activity. Consistent with a model of a negative EGFR regulation by RanBP6, we observed an inverse correlation between RanBP6 and EGFR mRNA levels in PTEN wildtype but not PTEN altered cancer cells in a large panel of human cancer cell lines (Cancer Cell Line Encyclopedia). To further understand the mechanism of how RanBP6 negatively regulates EGFR mRNA level, we found that RanBP6 interacted with nuclear Ran-GTPase and repressed EGFR transcription by promoting nuclear import of Signal transducer and activator of transcription 3 (STAT3). Lastly, RanBP6 appeared to be frequently deleted on chromosome 9p in GBM. We showed that RanBP6 silencing raised EGFR levels and signal output and accelerated in-vivo glioma growth. Our results establish a novel function of RanBP6 as a link between EGFR signaling and the Ran-mediated nuclear import pathway, and identify RanBP6 as candidate tumor suppressor on chromosome 9p. Citation Format: Wan-Ying Hsieh, Barbara Oldrini, Hediye Erdjument-Bromage, Paolo Codega, Maria S. Carro, Igor Vivanco, Dan Rohle, Carl Campos, Craig Bielski, Barry Taylor, Paul Tempst, Massimo Squatrito, Ingo K. Mellinghoff. Identification of Ran binding protein 6 as a novel negative regulator of EGFR and candidate tumor suppressor in glioblastoma [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 1032. doi:10.1158/1538-7445.AM2017-1032

PTEN mutations are among the most frequent genetic alterations found in human prostate cancers. Our previous works suggest that although precancerous lesions were found in Pten heterozygous mice, cancer progression and metastasis only happened when both alleles of Pten were deleted. To understand the molecular mechanisms underlying the role of PTEN in prostate cancer control, we generated two pairs of isogenic, androgen receptor (AR)–positive prostate epithelial lines from intact conditional Pten knock-out mice that are either heterozygous (PTEN-P2 and -P8) or homozygous (PTEN-CaP2 and PTEN-CaP8) for Pten deletion. Further characterization of these cells showed that loss of the second allele of Pten leads to increased anchorage-independent growth in vitro and tumorigenesis in vivo without obvious structural or numerical chromosome changes based on SKY karyotyping analysis. Despite no prior exposure to hormone ablation therapy, Pten null cells are tumorigenic in both male and female severe combined immunodeficiency mice. Furthermore, knocking down PTEN can convert the androgen-dependent Myc-CaP cell into androgen independence, suggesting that PTEN intrinsically controls androgen responsiveness, a critical step in the development of hormone refractory prostate cancer. Importantly, knocking down AR by shRNA in Pten null cells reverses androgen-independent growth in vitro and partially inhibited tumorigenesis in vivo, indicating that PTEN-controlled prostate tumorigenesis is AR dependent. These cell lines will serve as useful tools for understanding signaling pathways controlled by PTEN and elucidating the molecular mechanisms involved in hormone refractory prostate cancer formation. [Cancer Res 2007;67(13):6083–91]

The epidermal growth factor receptor (EGFR) is commonly amplified, overexpressed, and mutated in glioblastoma, making it a compelling molecular target for therapy. We have recently shown that coexpression of EGFRvIII and PTEN protein by glioblastoma cells is strongly associated with clinical response to EGFR kinase inhibitor therapy. PTEN loss, by dissociating inhibition of the EGFR from downstream phosphatidylinositol 3-kinase (PI3K) pathway inhibition, seems to act as a resistance factor. Because 40% to 50% of glioblastomas are PTEN deficient, a critical challenge is to identify strategies that promote responsiveness to EGFR kinase inhibitors in patients whose tumors lack PTEN. Here, we show that the mammalian target of rapamycin (mTOR) inhibitor rapamycin enhances the sensitivity of PTEN-deficient tumor cells to the EGFR kinase inhibitor erlotinib. In two isogenic model systems (U87MG glioblastoma cells expressing EGFR, EGFRvIII, and PTEN in relevant combinations, and SF295 glioblastoma cells in which PTEN protein expression has been stably restored), we show that combined EGFR/mTOR kinase inhibition inhibits tumor cell growth and has an additive effect on inhibiting downstream PI3K pathway signaling. We also show that combination therapy provides added benefit in promoting cell death in PTEN-deficient tumor cells. These studies provide strong rationale for combined mTOR/EGFR kinase inhibitor therapy in glioblastoma patients, particularly those with PTEN-deficient tumors. (Cancer Res 2006; 66(16): 7864-9)

We identified TMPRSS2 as a gene that is down-regulated in androgen-independent prostate cancer xenograft tissue derived from a bone metastasis. Using specific monoclonal antibodies, we show that the TMPRSS2-encoded serine protease is expressed as a Mr 70,000 full-length form and a cleaved Mr 32,000 protease domain. Mutation of Ser-441 in the catalytic triad shows that the proteolytic cleavage is dependent on catalytic activity, suggesting that it occurs as a result of autocleavage. Mutational analysis reveals the cleavage site to be at Arg-255. A consequence of autocatalytic cleavage is the secretion of the protease domain into the media by TMPRSS2-expressing prostate cancer cells and into the sera of prostate tumor-bearing mice. Immunohistochemical analysis of clinical specimens demonstrates the highest expression of TMPRSS2 at the apical side of prostate and prostate cancer secretory epithelia and within the lumen of the glands. Similar luminal staining was detected in colon cancer samples. Expression was also seen in colon and pancreas, with little to no expression detected in seven additional normal tissues. These data demonstrate that TMPRSS2 is a secreted protease that is highly expressed in prostate and prostate cancer, making it a potential target for cancer therapy and diagnosis.