The Dual PI3K/mTOR Inhibitor NVP-BEZ235 Induces Tumor Regression in a Genetically Engineered Mouse Model of PIK3CA Wild-Type Colorectal Cancer

To examine the in vitro and in vivo efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type colorectal cancer (CRC).PIK3CA mutant and wild-type human CRC cell lines were treated in vitro with NVP-BEZ235, and the resulting effects on proliferation, apoptosis, and signaling were assessed. Colonic tumors from a genetically engineered mouse (GEM) model for sporadic wild-type PIK3CA CRC were treated in vivo with NVP-BEZ235. The resulting effects on macroscopic tumor growth/regression, proliferation, apoptosis, angiogenesis, and signaling were examined.In vitro treatment of CRC cell lines with NVP-BEZ235 resulted in transient PI3K blockade, sustained decreases in mTORC1/mTORC2 signaling, and a corresponding decrease in cell viability (median IC(50) = 9.0-14.3 nM). Similar effects were seen in paired isogenic CRC cell lines that differed only in the presence or absence of an activating PIK3CA mutant allele. In vivo treatment of colonic tumor-bearing mice with NVP-BEZ235 resulted in transient PI3K inhibition and sustained blockade of mTORC1/mTORC2 signaling. Longitudinal tumor surveillance by optical colonoscopy demonstrated a 97% increase in tumor size in control mice (p = 0.01) vs. a 43% decrease (p = 0.008) in treated mice. Ex vivo analysis of the NVP-BEZ235-treated tumors demonstrated a 56% decrease in proliferation (p = 0.003), no effects on apoptosis, and a 75% reduction in angiogenesis (p = 0.013).These studies provide the preclinical rationale for studies examining the efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type CRC

Combination PI3K/MEK inhibition promotes tumor apoptosis and regression in PIK3CA wild-type, KRAS mutant colorectal cancer

PI3K inhibition in combination with other agents has not been studied in the context of PIK3CA wild-type, KRAS mutant cancer. In a screen of phospho-kinases, PI3K inhibition of KRAS mutant colorectal cancer cells activated the MAPK pathway. Combination PI3K/MEK inhibition with NVP-BKM120 and PD-0325901 induced tumor regression in a mouse model of PIK3CA wild-type, KRAS mutant colorectal cancer, which was mediated by inhibition of mTORC1, inhibition of MCL-1, and activation of BIM. These findings implicate mitochondrial-dependent apoptotic mechanisms as determinants for the efficacy of PI3K/MEK inhibition in the treatment of PIK3CA wild-type, KRAS mutant cancer. Keywords: PI3K; MEK; KRAS; Colorectal cancer; Mouse model of cance

Development of a mouse model for sporadic and metastatic colon tumors and its use in assessing drug treatment

Most genetically engineered mouse (GEM) models for colon cancer are based on tissuewide or germline gene modification, resulting in tumors predominantly of the small intestine. Several of these models involve modification of the adenomatous polyposis coli (Apc) gene and are excellent models for familial cancer predisposition syndromes. We have developed a stochastic somatic mutation model for sporadic colon cancer that presents with isolated primary tumors in the distal colon and recapitulates the entire adenoma–carcinoma–metastasis axis seen in human colon cancer. Using this model, we have analyzed tumors that are either solely mutant in the Apc gene or in combination with another colon cancer-associated mutant gene, the Kras G12D allele. Because of the restricted location in the distal colon, the natural history of the tumors can be analyzed by serial colonoscopy. As the mammalian target of rapamycin (mTOR) pathway is a critical component of the complex signaling network in colon cancer, we used this model to assess the efficacy of mTOR blockade through rapamycin treatment of mice with established tumors. After treatment, Apc mutant tumors were more than 80% smaller than control tumors. However, tumors that possessed both Apc and Kras mutations did not respond to rapamycin treatment. These studies suggest that mTOR inhibitors should be further explored as potential colorectal cancer therapies in patients whose tumors do not have activating mutations in KRAS

PKM2 is not required for colon cancer initiated by APC loss

Background: Cancer cells express the M2 isoform of the glycolytic enzyme pyruvate kinase (PKM2). PKM2 expression is not required for some cancers, and PKM2 loss can promote cancer progression; however, PKM2 has been reported to be essential in other tumor contexts, including a proposed non-metabolic role in β-catenin nuclear translocation. PKM2 is expressed in colon cancers where loss of the Apc tumor suppressor results in β-catenin nuclear translocation and aberrant activation of the canonical Wnt signaling pathway. Whether PKM2 is required in this colon cancer context has not been investigated. Results: Colon tumorigenesis was induced in mice harboring conditional Apc and Pkm2 alleles, and tumor progression was monitored by serial colonoscopy. PKM2 deletion had no effect on overall survival, the number of mice that developed tumors, or the number of tumors that developed per animal. Immunohistochemical analysis demonstrated PKM2 expression in wild-type tumors and the expected loss of PKM2 expression in tumors from Pkm2 conditional mice. Loss of PKM2 resulted in pyruvate kinase M1 expression but had no effect on nuclear β-catenin staining. These findings are consistent with tumor growth and activated Wnt signaling despite PKM2 loss in this model. We also found a large fraction of human colon cancers had very low or undetectable levels of PKM2 expression. Conclusions: PKM2 is not required for Apc-deficient colon cancer or for nuclear translocation of β-catenin in Apc-null tumor cells. These findings suggest that PKM2 expression is not required for colon tumor formation or progression. Electronic supplementary material The online version of this article (10.1186/s40170-017-0172-1) contains supplementary material, which is available to authorized users

<i>In vivo</i> NVP-BEZ235 treatment of a GEM model for sporadic CRC results in transient PI3K blockade inhibition and sustained mTORC1/mTORC2 blockade.

<p>Mice with colonic tumors were randomized to treatment with control diluent or 45 mg/kg NVP-BEZ235 by daily oral gavage for five days. Western blot analysis of p-AKT^Thr308, p-AKT^Ser473, and p-S6^Ser240/244 was performed for tumors treated with (−) control diluent or (+) NVP-BEZ235 for (A) five and (B) 28 days. (C) Immunohistochemistry of p-AKT^Ser473, p-S6^Ser240/244, and p-S6^Ser235/236 was performed for tumors treated with control diluent or NVP-BEZ235 for 28 days.</p

<i>In vivo</i> treatment of a GEM model for sporadic CRC results in tumor regression.

<p>Mice with colonic tumors were randomized to treatment with control diluent (N = 8) or 45 mg/kg NVP-BEZ235 (N = 8) by daily oral gavage for 28 days. Resulting colonic tumor growth or regression was serially examined by optical colonoscopy. (A) Tumor Size Index (TSI) was calculated as Tumor Area (T) divided by Lumen Area (L) ×100. (B) Representative tumor colonoscopy still images during the 28 day treatment period. (C) Tumor volume of control and NVP-BEZ235-treated tumors at necropsy (mean 65 mm³ vs. 5 mm³; p = 0.01). (D) Final TSI vs. Tumor Volume (R² = 0.89, P<0.0001). Change in mean TSI for (E) control (32% pre-treatment vs. 57% post-treatment, P = 0.01) and (F) treated (32% vs. 20%, P = 0.02) cohorts.</p

Effects of NVP-BEZ235 on cell viability and PI3K/mTOR signaling are independent of <i>PIK3CA</i> status.

<p>(A) Cell viability of mutant and wild-type isogenic <i>PIK3CA</i> cells was assessed by MTS assay after treatment with increasing concentrations (0–500 nM) of NVP-BEZ235 for 48 hours. Results shown are the mean of four independent experiments. (B) Western blot analysis for p-AKT^Thr308, p-AKT^Ser473, p-S6^Ser240/244, p-S6^Ser235/236, cleaved caspase 3, and cleaved PARP was performed after 2, 6, 24, and 48 hours incubation with (−) 0 or (+) 500 nM NVP-BEZ235.</p