Valentina Boscaro

Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer

A main limitation of therapies that selectively target kinase signalling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR), is effective in a subset of KRAS wild-type metastatic colorectal cancers. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood. Here we show that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance, but resistant cells remained sensitive to combinatorial inhibition of EGFR and mitogen-activated protein-kinase kinase (MEK). Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60% (6 out of 10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab-treated patients as early as 10 months before radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months before radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.

Molecular landscape of acquired resistance to targeted therapy combinations in BRAF mutant colorectal cancer

Although recent clinical trials of BRAF inhibitor combinations have demonstrated improved efficacy in BRAF-mutant colorectal cancer, emergence of acquired resistance limits clinical benefit. Here, we undertook a comprehensive effort to define mechanisms underlying drug resistance with the goal of guiding development of therapeutic strategies to overcome this limitation. We generated a broad panel of BRAF-mutant resistant cell line models across seven different clinically relevant drug combinations. Combinatorial drug treatments were able to abrogate ERK1/2 phosphorylation in parental-sensitive cells, but not in their resistant counterparts, indicating that resistant cells escaped drug treatments through one or more mechanisms leading to biochemical reactivation of the MAPK signaling pathway. Genotyping of resistant cells identified gene amplification of EGFR, KRAS, and mutant BRAF, as well as acquired mutations in KRAS, EGFR, and MAP2K1 These mechanisms were clinically relevant, as we identified emergence of a KRAS G12C mutation and increase of mutant BRAF V600E allele frequency in the circulating tumor DNA of a patient at relapse from combined treatment with BRAF and MEK inhibitors. To identify therapeutic combinations capable of overcoming drug resistance, we performed a systematic assessment of candidate therapies across the panel of resistant cell lines. Independent of the molecular alteration acquired upon drug pressure, most resistant cells retained sensitivity to vertical MAPK pathway suppression when combinations of ERK, BRAF, and EGFR inhibitors were applied. These therapeutic combinations represent promising strategies for future clinical trials in BRAF-mutant colorectal cancer. Cancer Res; 76(15); 4504-15. ©2016 AACR.

BRAF V600E Is a Determinant of Sensitivity to Proteasome Inhibitors

A critical step toward defining tailored therapy in patients with cancer is the identification of genetic interactions that may impair—or boost—the efficacy of selected therapeutic approaches. Cell models able to recapitulate combinations of genetic aberrations are important to find drug–genotype interactions poorly affected by the heterogeneous genetics of human tumors. In order to identify novel pharmacogenomic relationships, we employed an isogenic cell panel that reconstructs cancer genetic scenarios. We screened a library of 43 compounds in human hTERT-HME1 epithelial cells in which PTEN or RB1 were silenced in combination with the targeted knockin of cancer-associated mutations in EGFR, KRAS, BRAF, or PIK3CA oncogenes. Statistical analysis and clustering algorithms were applied to display similar drug response profiles and mutation-specific patterns of activity. From the screen, we discovered that proteasome inhibitors show selectivity toward BRAF V600E–mutant cells, irrespective of PTEN or RB1 expression. Preferential targeting of BRAF-mutant cells by proteasome inhibitors was corroborated in a second BRAF V600E isogenic model, as well as in a panel of colorectal cancer cell lines by the use of the proteasome inhibitor carfilzomib. Notably, carfilzomib also showed striking in vivo activity in a BRAF-mutant human colorectal cancer xenograft model. Vulnerability to proteasome inhibitors is dependent on persistent BRAF signaling, because BRAF V600E blockade by PLX4720 reversed sensitivity to carfilzomib in BRAF-mutant colorectal cancer cells. Our findings indicated that proteasome inhibition might represent a valuable targeting strategy in BRAF V600E–mutant colorectal tumors. Mol Cancer Ther; 12(12); 2950–61. ©2013 AACR.

SYNTHESIS, CHARACTERIZATION AND CELL VIABILITY TEST OF SIX VANADYL COMPLEXES WITH ACETYLACETONATE DERIVATIVES

Vanadium compounds are known to display a number of therapeutic effects, namely insulin-mimetic and cardiovascular effects. Evidence of the antiproliferative and proapoptotic activity of a number of vanadyl complexes, together with their low toxicity, establishes these metal compounds as promising antitumoral therapeutic agents. In the present work, we describe the synthesis and full characterization of six new vanadyl complexes with acetylacetonate derivatives bearing asymmetric substitutions on the β-dicarbonyl moiety: the complexes were characterized in the solid state as well as in solution. Our results show that all complexes are in square pyramidal geometry; cis isomers in the equatorial plane are favored in the presence of strongly coordinating solvents. EPR evidence suggests that all complexes are in the bis-chelate form, although in two cases the mono-chelated complex seems to be present as well. Preliminary tests carried out on non-tumor and tumor cell lines show that these complexes are effective in suppressing cell viability and elicit a distinct response of tumor and non-tumor cells.

Abstract 230: Modeling tumor progression and identifying genotype-drug interactions by the sequential introduction of cancer mutations into the genome of human normal cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The mutational landscape of cancer genomes displays a complex combination of genetic lesions affecting oncogenes and tumor suppressor genes. The construction of cellular models closely recapitulating cancer-linked genetic alterations is a prerequisite to understand their role in tumor progression and to identify genotype- specific pharmacological responses. To recapitulate point mutations affecting oncogenes we used homologous recombination to ‘knock-in’ specific nucleotide changes in the genome of human cells. We focused on the following alleles: EGFR (delE746-A750), KRAS (G13D), BRAF (V600E) and PIK3CA (E545K and H1047R), that are found in multiple human cancers. As recipient cells, a human non transformed epithelial cell line of breast origin (hTERT-HME1) has been employed. This cell line has been immortalized by hTERT and harbors a mutation on TP53 gene, a genetic event frequently observed in naturally occurring human tumors. To recapitulate the inactivation of additional tumor suppressor genes in our cellular models, we exploited shRNAs to permanently knock-down the expression of PTEN and RB1 genes, as they are frequently down regulated in human tumors. Knock-down of PTEN and RB1 was performed both in the parental hTERT-HME1 cells and in the ‘knock-in’ models, thus allowing us to combine tumor suppressor genes inactivation with mutational activation of specific oncogenes. The resulting cellular models are hereafter refereed to as combinatorial ‘matrix’. To assess how specific combinations of genetic lesions affect the tumorigenic properties of not transformed cells, we measured the ability of the ‘matrix’ to grow in soft agar. At the same time we injected our cellular models in the cleared, humanized mammary fat pad of immunocompromised mice. For the latter approach, we focused on genotypes recapitulating mutations found in breast tumors, such as PTEN or RB1 inactivation and PIK3CA or KRAS mutations, alone or in combination. In vitro and in vivo results showed that those combinations of genetic events are not sufficient to transform the normal hTERT-HME1 cells. However, preliminary data indicates that few genotypes of the ‘matrix’ acquired the ability to colonize the murine fat pads. With the rational of unveiling new pharmaco-genetic interactions involving one or multiple cancer mutations, the “matrix” was used to screen a panel of compounds used for cancer therapy, or for which an anti-proliferative activity was previously reported. This approach led to the identification of a subset of genotype-drug interactions thus underscoring how the multifactorial genomic milieu of cancer cells impact the response to therapies. Grant support: This work is part of the EU FP7 grant COLTHERES Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 230. doi:10.1158/1538-7445.AM2011-230