Anna Staniszewska

Targeting KRAS-dependent tumors with AZD4785, a high-affinity therapeutic antisense oligonucleotide inhibitor of KRAS

AZD4785 is a therapeutic antisense oligonucleotide targeting KRAS mRNA with promising preclinical antitumor activity and a favorable safety profile. An antisensible approach to targeting KRAS Mutations that cause activation of the KRAS oncogene are common in human cancer, including treatment-resistant tumor types such as lung and pancreatic cancer. KRAS has also proven to be notoriously difficult to target with small molecules. To overcome this issue, Ross et al. have turned to genetic technology, demonstrating an antisense oligonucleotide–based therapy for inhibiting KRAS. The antisense oligonucleotide used in this study was chemically modified, allowing systemic delivery through subcutaneous injection and avoiding the need for a specialized delivery vehicle. The authors tested the efficacy of this therapy in multiple mouse models of non–small cell lung cancer and evaluated its safety in primates, demonstrating its potential suitability for translation to humans. Activating mutations in KRAS underlie the pathogenesis of up to 20% of human tumors, and KRAS is one of the most frequently mutated genes in cancer. Developing therapeutics to block KRAS activity has proven difficult, and no direct inhibitor of KRAS function has entered clinical trials. We describe the preclinical evaluation of AZD4785, a high-affinity constrained ethyl–containing therapeutic antisense oligonucleotide (ASO) targeting KRAS mRNA. AZD4785 potently and selectively depleted cellular KRAS mRNA and protein, resulting in inhibition of downstream effector pathways and antiproliferative effects selectively in KRAS mutant cells. AZD4785-mediated depletion of KRAS was not associated with feedback activation of the mitogen-activated protein kinase (MAPK) pathway, which is seen with RAS-MAPK pathway inhibitors. Systemic delivery of AZD4785 to mice bearing KRAS mutant non–small cell lung cancer cell line xenografts or patient-derived xenografts resulted in inhibition of KRAS expression in tumors and antitumor activity. The safety of this approach was demonstrated in mice and monkeys with KRAS ASOs that produced robust target knockdown in a broad set of tissues without any adverse effects. Together, these data suggest that AZD4785 is an attractive therapeutic for the treatment of KRAS-driven human cancers and warrants further development.

Antitumor Activity of Osimertinib, an Irreversible Mutant-Selective EGFR Tyrosine Kinase Inhibitor, in NSCLC Harboring EGFR Exon 20 Insertions.

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.

Combined inhibition of mTOR and CDK4/6 is required for optimal blockade of E2F function and long term growth inhibition in estrogen receptor positive breast cancer

The cyclin dependent kinase (CDK)–retinoblastoma (RB)–E2F pathway plays a critical role in the control of cell cycle in estrogen receptor–positive (ER+) breast cancer. Small-molecule inhibitors of CDK4/6 have shown promise in this tumor type in combination with hormonal therapies, reflecting the particular dependence of this subtype of cancer on cyclin D1 and E2F transcription factors. mTOR inhibitors have also shown potential in clinical trials in this disease setting. Recent data have suggested cooperation between the PI3K/mTOR pathway and CDK4/6 inhibition in preventing early adaptation and eliciting growth arrest, but the mechanisms of the interplay between these pathways have not been fully elucidated. Here we show that profound and durable inhibition of ER+ breast cancer growth is likely to require multiple hits on E2F-mediated transcription. We demonstrate that inhibition of mTORC1/2 does not affect ER function directly, but does cause a decrease in cyclin D1 protein, RB phosphorylation, and E2F-mediated transcription. Combination of an mTORC1/2 inhibitor with a CDK4/6 inhibitor results in more profound effects on E2F-dependent transcription, which translates into more durable growth arrest and a delay in the onset of resistance. Combined inhibition of mTORC1/2, CDK4/6, and ER delivers even more profound and durable regressions in breast cancer cell lines and xenografts. Furthermore, we show that CDK4/6 inhibitor–resistant cell lines reactivate the CDK–RB–E2F pathway, but remain sensitive to mTORC1/2 inhibition, suggesting that mTORC1/2 inhibitors may represent an option for patients that have relapsed on CDK4/6 therapy. Mol Cancer Ther; 17(5); 908–20. ©2018 AACR.

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.

Combination of dual mTORC1/2 inhibition and immune-checkpoint blockade potentiates anti-tumour immunity

ABSTRACT mTOR inhibition can promote or inhibit immune responses in a context dependent manner, but whether this will represent a net benefit or be contraindicated in the context of immunooncology therapies is less understood. Here, we report that the mTORC1/2 dual kinase inhibitor vistusertib (AZD2014) potentiates anti-tumour immunity in combination with anti-CTLA-4 (αCTLA-4), αPD-1 or αPD-L1 immune checkpoint blockade. Combination of vistusertib and immune checkpoint blocking antibodies led to tumour growth inhibition and improved survival of MC-38 or CT-26 pre-clinical syngeneic tumour models, whereas monotherapies were less effective. Underlying these combinatorial effects, vistusertib/immune checkpoint combinations reduced the occurrence of exhausted phenotype tumour infiltrating lymphocytes (TILs), whilst increasing frequencies of activated Th1 polarized T-cells in tumours. Vistusertib alone was shown to promote a Th1 polarizing proinflammatory cytokine profile by innate primary immune cells. Moreover, vistusertib directly enhanced activation of effector T-cell and survival, an effect that was critically dependent on inhibitor dose. Therefore, these data highlight direct, tumour-relevant immune potentiating benefits of mTOR inhibition that complement immune checkpoint blockade. Together, these data provide a clear rationale to investigate such combinations in the clinic.

Abstract 2079: Osimertinib, an irreversible mutant selective EGFR tyrosine kinase inhibitor, exerts anti tumor activity in NSCLC harbouring exon 20 insertion mutant-EGFR

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 Floc9h, 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 O9Neill, 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

Abstract B155: VER-246608, a novel pan-isoform ATP competitive inhibitor of pyruvate dehydrogenase kinase, disrupts Warburg metabolism and demonstrates context-dependent cytotoxicity to cancer cells.

Pyruvate dehydrogenase kinase (PDK) regulates the activity of the pyruvate dehydrogenase complex (PDC) through phosphorylation of three serine residues on the E1α subunit, resulting in decreased activity. The expression of all four mammalian isoforms of PDK have been shown to be up-regulated either under tumour relevant conditions (PDK-1 & PDK-3 by hypoxia) or by the loss of function of common tumour suppressor genes (PDK-2 by p53; PDK-4 by pRB). Furthermore, PDK-1 expression has been clinically correlated with poor prognosis in Gastric, HNSCC and Colon cancer. Previous studies employing RNAi have provided evidence for a survival role for PDK-1 as well as its importance in maintaining the glycolytic phenotype of cancer cells. DCA, a weak inhibitor of PDK, has also been used to study the role of PDK in cancer; however, interpretation of these studies has been complicated by conflicting data and the lack of specificity of this agent for PDK. Here we report the discovery of a novel, potent and selective pan-isoform inhibitor of PDK, VER-246608. Consistent with a PDK mediated MOA, treatment of PC-3 cells with VER-246608 resulted in increased PDC activity and oxygen consumption as well as reduced lactate production and glucose consumption. Interestingly, modulation of glycolytic activity required compound concentrations which achieved > 90 % reduction in E1α phosphorylation and this was only observed under glucose depleted conditions. Under normal culture conditions, VER-246608 showed little cytotoxicity to cancer cells. We hypothesised that the supraphysiological glucose concentrations present in cell culture media may limit the effect of PDK inhibition due to elevated intracellular pyruvate levels. We therefore performed cytotoxicity studies under conditions of limited nutrient availability. We found that combined depletion of glucose and glutamine or serum alone resulted in enhanced cytotoxicity vs normal media; however, hypoxic conditions had no effect. Furthermore, this differential cytotoxicity correlated with reduced pyruvate levels in the compound treated cells cultured in the above ‘austere’ conditions. VER-246608 also inhibited the growth of tumour spheroids with a potency that was comparable to cells grown in 2D culture. In addition, combination treatment studies revealed that VER-246608 potentiated anti-cancer agents which are known to influence mitochondrial function. In contrast, the lipoamide binding site inhibitor, Nov3r, showed no evidence of cytotoxicity under any of the above conditions and was ineffective in altering glycolytic activity. These studies suggest that PDK inhibition may be effective under the nutrient depleted conditions found in the tumour microenvironment and that combination treatments should be explored to reveal the full potential of this therapeutic strategy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B155. Citation Format: Jonathan Moore, Anna Staniszewska, Terence Shaw, Jalanie D9Alessandro, Ben Davis, Alan Surgenor, Lisa Baker, Natalia Massanova, James Murray, Alba Macias, Paul Brough, Mike Wood, Patrick C. Mahon. VER-246608, a novel pan-isoform ATP competitive inhibitor of pyruvate dehydrogenase kinase, disrupts Warburg metabolism and demonstrates context-dependent cytotoxicity to cancer cells. [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 B155.