Marie Cumberbatch

AZD3514: A Small Molecule That Modulates Androgen Receptor Signaling and Function In Vitro and In Vivo

Continued androgen receptor (AR) expression and signaling is a key driver in castration-resistant prostate cancer (CRPC) after classical androgen ablation therapies have failed, and therefore remains a target for the treatment of progressive disease. Here, we describe the biological characterization of AZD3514, an orally bioavailable drug that inhibits androgen-dependent and -independent AR signaling. AZD3514 modulates AR signaling through two distinct mechanisms, an inhibition of ligand-driven nuclear translocation of AR and a downregulation of receptor levels, both of which were observed in vitro and in vivo. AZD3514 inhibited testosterone-driven seminal vesicle development in juvenile male rats and the growth of androgen-dependent Dunning R3327H prostate tumors in adult rats. Furthermore, this class of compound showed antitumor activity in the HID28 mouse model of CRPC in vivo. AZD3514 is currently in phase I clinical evaluation. Mol Cancer Ther; 12(9); 1715–27. ©2013 AACR.

Inhibition of PI3Kβ Signaling with AZD8186 Inhibits Growth of PTEN-Deficient Breast and Prostate Tumors Alone and in Combination with Docetaxel

Loss of PTEN protein results in upregulation of the PI3K/AKT pathway, which appears dependent on the PI3Kβ isoform. Inhibitors of PI3Kβ have potential to reduce growth of tumors in which loss of PTEN drives tumor progression. We have developed a small-molecule inhibitor of PI3Kβ and PI3Kδ (AZD8186) and assessed its antitumor activity across a panel of cell lines. We have then explored the antitumor effects as single agent and in combination with docetaxel in triple-negative breast (TNBC) and prostate cancer models. In vitro, AZD8186 inhibited growth of a range of cell lines. Sensitivity was associated with inhibition of the AKT pathway. Cells sensitive to AZD8186 (GI50 < 1 μmol/L) are enriched for, but not exclusively associated with, PTEN deficiency. In vivo, AZD8186 inhibits PI3K pathway biomarkers in prostate and TNBC tumors. Scheduling treatment with AZD8186 shows antitumor activity required only intermittent exposure, and that increased tumor control is achieved when AZD8186 is used in combination with docetaxel. AZD8186 is a potent inhibitor of PI3Kβ with activity against PI3Kδ signaling, and has potential to reduce growth of tumors dependent on dysregulated PTEN for growth. Moreover, AZD8186 can be combined with docetaxel, a chemotherapy commonly used to treat advanced TBNC and prostate tumors. The ability to schedule AZD8186 and maintain efficacy offers opportunity to combine AZD8186 more effectively with other drugs. Mol Cancer Ther; 14(1); 48–58. ©2014 AACR.

Identification of a Subset of Human Non–Small Cell Lung Cancer Patients with High PI3Kβ and Low PTEN Expression, More Prevalent in Squamous Cell Carcinoma

Purpose: The phosphoinositide 3-kinase (PI3K) pathway is a major oncogenic signaling pathway and an attractive target for therapeutic intervention. Signaling through the PI3K pathway is moderated by the tumor suppressor PTEN, which is deficient or mutated in many human cancers. Molecular characterization of the PI3K signaling network has not been well defined in lung cancer; in particular, the role of PI3Kβ and its relation to PTEN in non–small cell lung cancer NSCLC remain unclear. Experimental Design: Antibodies directed against PI3Kβ and PTEN were validated and used to examine, by immunohistochemistry, expression in 240 NSCLC resection tissues [tissue microarray (TMA) set 1]. Preliminary observations were extended to an independent set of tissues (TMA set 2) comprising 820 NSCLC patient samples analyzed in a separate laboratory applying the same validated antibodies and staining protocols. The staining intensities for PI3Kβ and PTEN were explored and colocalization of these markers in individual tumor cores were correlated. Results: PI3Kβ expression was elevated significantly in squamous cell carcinomas (SCC) compared with adenocarcinomas. In contrast, PTEN loss was greater in SCC than in adenocarcinoma. Detailed correlative analyses of individual patient samples revealed a significantly greater proportion of SCC in TMA set 1 with higher PI3Kβ and lower PTEN expression when compared with adenocarcinoma. These findings were reinforced following independent analyses of TMA set 2. Conclusions: We identify for the first time a subset of NSCLC more prevalent in SCC, with elevated expression of PI3Kβ accompanied by a reduction/loss of PTEN, for whom selective PI3Kβ inhibitors may be predicted to achieve greater clinical benefit. Clin Cancer Res; 20(3); 595–603. ©2013 AACR.

Intermittent High-Dose Scheduling of AZD8835, a Novel Selective Inhibitor of PI3Kα and PI3Kδ, Demonstrates Treatment Strategies for PIK3CA-Dependent Breast Cancers

The PIK3CA gene, encoding the p110α catalytic unit of PI3Kα, is one of the most frequently mutated oncogenes in human cancer. Hence, PI3Kα is a target subject to intensive efforts in identifying inhibitors and evaluating their therapeutic potential. Here, we report studies with a novel PI3K inhibitor, AZD8835, currently in phase I clinical evaluation. AZD8835 is a potent inhibitor of PI3Kα and PI3Kδ with selectivity versus PI3Kβ, PI3Kγ, and other kinases that preferentially inhibited growth in cells with mutant PIK3CA status, such as in estrogen receptor–positive (ER+) breast cancer cell lines BT474, MCF7, and T47D (sub-μmol/L GI50s). Consistent with this, AZD8835 demonstrated antitumor efficacy in corresponding breast cancer xenograft models when dosed continuously. In addition, an alternative approach of intermittent high-dose scheduling (IHDS) was explored given our observations that higher exposures achieved greater pathway inhibition and induced apoptosis. Indeed, using IHDS, monotherapy AZD8835 was able to induce tumor xenograft regression. Furthermore, AZD8835 IHDS in combination with other targeted therapeutic agents further enhanced antitumor activity (up to 92% regression). Combination partners were prioritized on the basis of our mechanistic insights demonstrating signaling pathway cross-talk, with a focus on targeting interdependent ER and/or CDK4/6 pathways or alternatively a node (mTOR) in the PI3K-pathway, approaches with demonstrated clinical benefit in ER+ breast cancer patients. In summary, AZD8835 IHDS delivers strong antitumor efficacy in a range of combination settings and provides a promising alternative to continuous dosing to optimize the therapeutic index in patients. Such schedules merit clinical evaluation. Mol Cancer Ther; 15(5); 877–89. ©2016 AACR.

The MEK inhibitor selumetinib complements CTLA-4 blockade by reprogramming the tumor immune microenvironment

Background T-cell checkpoint blockade and MEK inhibitor combinations are under clinical investigation. Despite progress elucidating the immuno-modulatory effects of MEK inhibitors as standalone therapies, the impact of MEK inhibition on the activity of T-cell checkpoint inhibitors remains incompletely understood. Here we sought to characterize the combined effects of MEK inhibition and anti-CTLA-4 mAb (anti-CTLA-4) therapy, examining effects on both T-cells and tumor microenvironment (TME). Methods In mice, the effects of MEK inhibition, via selumetinib, and anti-CTLA-4 on immune responses to keyhole limpet haemocyanin (KLH) immunization were monitored using ex vivo functional assays with splenocytes. In a KRAS-mutant CT26 mouse colorectal cancer model, the impact on the tumor microenvironment (TME) and the spleen were evaluated by flow cytometry. The TME was further examined by gene expression and immunohistochemical analyses. The combination and sequencing of selumetinib and anti-CTLA-4 were also evaluated in efficacy studies using the CT26 mouse syngeneic model. Results Anti-CTLA-4 enhanced the generation of KLH specific immunity following KLH immunization in vivo; selumetinib was found to reduce, but did not prevent, this enhancement of immune response by anti-CTLA-4 in vivo. In the CT26 mouse model, anti-CTLA-4 treatment led to higher expression levels of the immunosuppressive mediators, Cox-2 and Arg1 in the TME. Combination of anti-CTLA-4 with selumetinib negated this up-regulation of Cox-2 and Arg1, reduced the frequency of CD11+ Ly6G+ myeloid cells, and led to the accumulation of differentiating monocytes at the Ly6C+ MHC+ intermediate state in the tumor. We also report that MEK inhibition had limited impact on anti-CTLA-4-mediated increases in T-cell infiltration and T-cell activation in CT26 tumors. Finally, we show that pre-treatment, but not concurrent treatment, with selumetinib enhanced the anti-tumor activity of anti-CTLA-4 in the CT26 model. Conclusion These data provide evidence that MEK inhibition can lead to changes in myeloid cells and immunosuppressive factors in the tumor, thus potentially conditioning the TME to facilitate improved response to anti-CTLA-4 treatment. In summary, the use of MEK inhibitors to alter the TME as an approach to enhance the activities of immune checkpoint inhibitors warrants further investigation in clinical trials. Electronic supplementary material The online version of this article (doi:10.1186/s40425-017-0268-8) contains supplementary material, which is available to authorized users.BackgroundT-cell checkpoint blockade and MEK inhibitor combinations are under clinical investigation. Despite progress elucidating the immuno-modulatory effects of MEK inhibitors as standalone therapies, the impact of MEK inhibition on the activity of T-cell checkpoint inhibitors remains incompletely understood. Here we sought to characterize the combined effects of MEK inhibition and anti-CTLA-4 mAb (anti-CTLA-4) therapy, examining effects on both T-cells and tumor microenvironment (TME).MethodsIn mice, the effects of MEK inhibition, via selumetinib, and anti-CTLA-4 on immune responses to keyhole limpet haemocyanin (KLH) immunization were monitored using ex vivo functional assays with splenocytes. In a KRAS-mutant CT26 mouse colorectal cancer model, the impact on the tumor microenvironment (TME) and the spleen were evaluated by flow cytometry. The TME was further examined by gene expression and immunohistochemical analyses. The combination and sequencing of selumetinib and anti-CTLA-4 were also evaluated in efficacy studies using the CT26 mouse syngeneic model.ResultsAnti-CTLA-4 enhanced the generation of KLH specific immunity following KLH immunization in vivo; selumetinib was found to reduce, but did not prevent, this enhancement of immune response by anti-CTLA-4 in vivo. In the CT26 mouse model, anti-CTLA-4 treatment led to higher expression levels of the immunosuppressive mediators, Cox-2 and Arg1 in the TME. Combination of anti-CTLA-4 with selumetinib negated this up-regulation of Cox-2 and Arg1, reduced the frequency of CD11+ Ly6G+ myeloid cells, and led to the accumulation of differentiating monocytes at the Ly6C+ MHC+ intermediate state in the tumor. We also report that MEK inhibition had limited impact on anti-CTLA-4-mediated increases in T-cell infiltration and T-cell activation in CT26 tumors. Finally, we show that pre-treatment, but not concurrent treatment, with selumetinib enhanced the anti-tumor activity of anti-CTLA-4 in the CT26 model.ConclusionThese data provide evidence that MEK inhibition can lead to changes in myeloid cells and immunosuppressive factors in the tumor, thus potentially conditioning the TME to facilitate improved response to anti-CTLA-4 treatment. In summary, the use of MEK inhibitors to alter the TME as an approach to enhance the activities of immune checkpoint inhibitors warrants further investigation in clinical trials.

Abstract 3264: AZD8186: a potent selective inhibitor of PI3Kβ targeting PTEN-deficient tumours dependent on dysregulated PI3Kβ signalling.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC AZD8186 is a novel potent small molecule that targets the lipid kinase PI3Kβ with selectivity vs PI3Kα. AZD8186 reduces pAKT-S473 in the PTEN deficient MDA-MB-468 cell line with an IC50 <5nM, while in the PI3Kα-dependent PIK3CA mutant line BT474 it is 200 fold less potent. AZD8186 also demonstrates cellular activity versus PI3Kβ, inhibiting IgM-dependent pAKT-S473 with an IC50 of 15nM. In cell line panels, sensitivity to AZD8186 is associated in part with loss of PTEN function suggesting that targeting tumours that have lost normal levels of PTEN function through deletion, mutation or down regulation might enrich for sensitivity to AZD8186. Deficiencies in PTEN are described in a number of tumour types such as prostate, squamous lung, breast, renal and glioblastoma. AZD8186 has single anti-tumour activity in pre-clinical models representing each of these settings, which is associated with dynamic regulation of a number of key pathway biomarkers. Clinically, agents such as AZD8186 are likely to be used in combination. The use of AZD8186 in conjunction with either androgen therapy or docetaxol has been explored in cell lines and pre-clinical disease models. Combining AZD8186 with MDV-3100 resulted in synergistic inhibition of LNCAP prostate tumour cell growth, demonstrating the potential to combine with androgen therapy. Moreover in HCC70 (breast) and PC3 (prostate) tumour xenografts AZD8186 shows increased benefit in combination with docetaxel, demonstrating the ability to combine with cytotoxic chemotherapy. Exploration of dose and schedule revealed that efficacy can be maintained in combination with docetaxol at a lower dose of AZD8186 than required for monotherapy activity. Moreover efficacy was maintained when the frequency of AZD8186 dosing was reduced using a shorter intermittent schedule. Understanding how AZD8186 combines with standards of care for PTEN null disease segments provides important insight into how agents targeting PI3Kβ-dependent tumours may be used most effectively in the clinic. [Current affiliation for S. Cosulich is Novartis, Basel, Switzerland.] Citation Format: Urs Hancox, Sabina Cosulich, Hannah Dry, Lynsey Hanson, Clare Crafter, Bernard Barlaam, Martina Fitzek, Lara Ward, Marie Cumberbatch, Steve Powell, Rebecca Ellston, Mandy Lawson, Steve Wedge, Liz Harrington, Stephen Green, Simon T. Barry. AZD8186: a potent selective inhibitor of PI3Kβ targeting PTEN-deficient tumours dependent on dysregulated PI3Kβ signalling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3264. doi:10.1158/1538-7445.AM2013-3264

Inhibiting PI3Kβ with AZD8186 Regulates Key Metabolic Pathways in PTEN-Null Tumors

Purpose: PTEN-null tumors become dependent on the PI3Kβ isoform and can be targeted by molecules such as the selective PI3Kβ inhibitor AZD8186. However, beyond the modulation of the canonical PI3K pathway, the consequences of inhibiting PI3Kβ are poorly defined. Experimental Design: To determine the broader impact of AZD8186 in PTEN-null tumors, we performed a genome-wide RNA-seq analysis of PTEN-null triple-negative breast tumor xenografts treated with AZD8186. Mechanistic consequences of AZD8186 treatment were examined across a number of PTEN-null cell lines and tumor models. Results: AZD8186 treatment resulted in modification of transcript and protein biomarkers associated with cell metabolism. We observed downregulation of cholesterol biosynthesis genes and upregulation of markers associated with metabolic stress. Downregulation of cholesterol biosynthesis proteins, such as HMGCS1, occurred in PTEN-null cell lines and tumor xenografts sensitive to AZD8186. Therapeutic inhibition of PI3Kβ also upregulated PDHK4 and increased PDH phosphorylation, indicative of reduced carbon flux into the TCA cycle. Consistent with this, metabolomic analysis revealed a number of changes in key carbon pathways, nucleotide, and amino acid biosynthesis. Conclusions: This study identifies novel mechanistic biomarkers of PI3Kβ inhibition in PTEN-null tumors supporting the concept that targeting PI3Kβ may exploit a metabolic dependency that contributes to therapeutic benefit in inducing cell stress. Considering these additional pathways will guide biomarker and combination strategies for this class of agents. Clin Cancer Res; 23(24); 7584–95. ©2017 AACR.

Abstract 2665: High dose intermittent scheduling of AZD8835, a novel potent and selective inhibitor of PI3Kα and PI3Kδ, identifies potential treatment strategies for PIK3CA-dependent cancers

The PIK3CA gene, encoding the p110 catalytic unit of PI3Kα, is one of the most frequently mutated oncogenes described in human cancer. Hence PI3Kα is a target subject to intensive efforts in identifying inhibitors and evaluating their therapeutic potential. To date most studies with PI3K inhibitors have used a continuous (daily) dosing schedule and although clinical responses have been reported the overall activity observed has been moderate. This may in part be due to suboptimal pathway inhibition which is capped by normal tissue toxicities such as rash, diarrhoea and hyperglycaemia. Furthermore, additional dose reduction may be required when such agents are used in combination with other therapies. Therefore in our efforts to optimise inhibition of PI3K pathway signalling, we have explored high dose intermittent scheduling as an alternative to continuous dosing. Here we describe pre-clinical studies that exemplify such concepts, centred around use of AZD8835, a PI3K inhibitor currently in Phase 1 clinical evaluation. AZD8835 is a novel and potent inhibitor of PI3Kα and PI3Kδ, with selectivity vs. PI3Kβ, PI3Kγ(IC50s of 6nM, 6nM, 431nM and 90nM respectively in enzyme assays) and other kinases. AZD8835 preferentially displays activity in tumour models with a mutant PIK3CA background, such as ER+ve breast cancer models. Such models were used in our investigations, in both cell culture and in mouse xenograft contexts. We demonstrate that a high dose intermittent schedule of single agent AZD8835 achieves greater pathway inhibition yielding significant anti-tumour responses. In the sensitive BT474 xenograft model, a dose of 100mg/kg AZD8835 BID on days 1 and 4 in a weekly schedule delivered -36% tumour regression, accompanied by a strongly elevated rapid onset apoptosis signal with 4-16% cells staining positively for cleaved-caspase3. We also evaluated AZD8835 in combination with other targeted therapeutic agents, in MCF7, BT474 and T47D breast models, observing increased sensitivity relative to single agent AZD8835; firstly with agents that target other nodes in the PI3K pathway; secondly with agents targeting parallel but interconnected driver pathways in breast disease (ER, CDK4/6). Overall the data indicate that high dose intermittent scheduling can deliver strong anti-tumour efficacy in a range of combination settings and provides a promising alternative to continuous dosing. Such schedules merit clinical evaluation. Citation Format: Kevin Hudson, Urs Hancox, Cath Trigwell, Phillippa Dudley, Lyndsey Hanson, Robert McEwen, Alys Jones, Marie Cumberbatch, Urszula Polanska, Rebecca Ellston, Oona Delpuech, Pablo Morentin Gutierrez, Lara Ward, Francisco Cruzalegui, Stephen Green. High dose intermittent scheduling of AZD8835, a novel potent and selective inhibitor of PI3Kα and PI3Kδ, identifies potential treatment strategies for PIK3CA-dependent cancers. [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 2665. doi:10.1158/1538-7445.AM2015-2665

Abstract 2668: Applying mechanistic PK/PD modeling to describe the efficacy of AZD8835 (PI3Kalpha/delta inhibitor) in a mouse xenograft breast tumor model at different dosing schedules

Introduction: PI3Kα inhibitors have shown antitumor efficacy in PIK3CA-mutant xenograft models when continued inhibition of the pathway is maintained (Fritsch et al., Mol Cancer Ther 2014; 13:1117-1129). The level of efficacy can be linked to the amount and duration of pathway inhibition using a proximal biomarker (pAKT). AZD8835 (a selective PI3Kα/δ inhibitor) delivers antitumor efficacy in a mouse xenografted breast cancer cell line (BT474c) using either a continuous or intermittent dosing schedule. However, the efficacy observed with the intermittent dosing schedule cannot be predicted based on the changes in the proximal biomarker alone. We present a novel mathematical model that links the efficacy observed with both schedules to the drug concentrations in plasma via drug-induced changes in the proximal biomarker (pAKT) as well as in a marker of apoptosis (Cleaved Caspase 3, CC3). Methods: All in vivo work was carried out in the mouse BT474c xenograft model. A mathematical pharmacokinetic/pharmacodynamic (PK/PD) model was built to link plasma concentrations of AZD8835 to both the proximal biomarker of pathway inhibition pAKT (measured using Western blot) and a distal marker of apoptosis, CC3 (measured by IHC). The output (CC3 and pAKT) of this model was used to drive an adaptation of a well-known model for tumor growth inhibition (Simeoni et al. Cancer Res 2004; 64:1094-1101). All doses and schedules were fitted simultaneously in a population approach. Results: The PK/PD model described well the time course of both biomarkers (pAKT and CC3), for a range of both continuous and intermittent doses. The CC3 PK/PD model captured the observation that on continuous dosing the CC3 signal diminishes while the signal can be maintained with intermittent dosing. By incorporating the time course of change in pAKT and CC3, the proposed model was able to simultaneously describe the effects of the different AZD8835 doses and schedules (continuous and intermittent) on tumor volume. Conclusions: Incorporating more mechanistic features to the drug-induced tumor growth inhibition model provides valuable insight to elucidate the relative balance of the anti-proliferative and pro-apoptotic consequences of PI3Kα/δ inhibition in a xenografted breast cancer cell line. This balance is dependent not only on the dose but also on the schedule. The model supports exploration of intermittent dosing in the clinic to help manage the chronic toxicology associated with PI3K pathway inhibition. Citation Format: Pablo Morentin Gutierrez, Kevin Hudson, Urs J. Hancox, Lara T. Ward, Urszula M. Polanska, Marie Cumberbatch, Francisco H. Cruzalegui. Applying mechanistic PK/PD modeling to describe the efficacy of AZD8835 (PI3Kalpha/delta inhibitor) in a mouse xenograft breast tumor model at different dosing schedules. [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 2668. doi:10.1158/1538-7445.AM2015-2668

Precise determination of toll-like receptor (TLR) 7 expression in multiple human tumor types

TLR7 agonists are being progressed as potential immunotherapeutics for the treatment of cancer. TLR7 agonism is believed to trigger a plasmacytoid dendritic cell driven immune response which drives anti-tumor efficacy. In addition to modulation of the immune system, TLR7 may be involved in tumor progression due to expression of TLR7 by tumor cells themselves [1]. To determine the precise distribution of TLR7 in different tumor types, a comprehensive validation of anti-TLR7 antibodies was conducted by immuohistochemistry (IHC). One antibody (Epitomics, 3923-1), out of five selected from scientific citations, proved specific for TLR7 following analyses by western blot of cell lysates, and by IHC of formalin fixed paraffin embedded (FFPE) cell pellets, prepared from HEK293 cells either stably transfected, mock-transfected or non-transfected with TLR7. 3923-1 was validated further across human spleen, lymph node and tonsil, revealing the expected tissue and cellular localization for these lymphoid organs. Assessment of TLR7 expression in a selection of tumor samples revealed non-specific tumor cell staining for the rejected antibodies, compared with minimal tumor staining for 3923-1. To explore further the distribution of TLR7, 5 tissue microarrays comprising 18 different human tumor types (6-25 patients/tumor type, triplicate cores) and 14 normal tissues (5 donors/tissue type, duplicate cores) were examined by IHC using 3923-1. Staining for TLR7 was scored by a pathologist (4-point scale: 0 negative, 1+ weak, 2+ moderate, 3+ strong) for tumor and immune cell compartments. Out of 18 tumor types examined, 5 were negative for tumor cell expression of TLR7 (ovarian, glioma, thyroid, liver, renal) and 9 exhibited a proportion (4%-36%) of patients with weak staining (breast, lung, colorectal, pancreatic, gastric, head & neck, melanoma, esophageal, endometrial). Moderate staining was observed for 11%-17% of sarcoma, prostate and bladder tumors. Corresponding normal tissue epithelium was largely negative for TLR7. Importantly, an increased density of immune infiltrates was observed in tumor tissues compared with normal tissues, and a greater proportion of the immune infiltrates were TLR7 positive. These data demonstrate that TLR7 may be less frequently expressed by tumor cells than suggested by the literature and that all tumor types exhibit a marked TLR7 positive immune cell infiltrate. Together, these data identify tumour types that might benefit from TLR7 therapy and may guide patient selection.