Mechanisms of synergistic suppression of ALK-positive lung cancer cell growth by the combination of ALK and SHP2 inhibitors.

Lung cancer is a major cause of cancer-related deaths. Alectinib is the first line of treatment for patients with ALK-positive lung cancer, but the survival rate beyond 2-3 years is low. Co-targeting secondary oncogenic drivers such as SHP2 is a potential strategy for improving drug efficacy. This is because SHP2 is expressed ubiquitously, but ALK expression is largely restricted to cancer cells. Thus, the combination of ALK and SHP2 inhibitors may provide a way to restrict synergistic cytotoxicity to cancer cells only, by reducing the dose of SHP2 inhibitors required for anticancer action and minimising SHP2-dependent systemic toxicity. The objective of this study was to investigate whether the combination of a SHP2 inhibitor (SHP099) with alectinib would synergistically suppress the growth of ALK-positive lung cancer cells. Our results demonstrated that the drug combination significantly and synergistically decreased cell viability at relatively low concentrations in ALK-positive H3122 and H2228 cells, due to G1 cell cycle arrest and increased apoptosis because of suppressed downstream RAS/MAPK signalling. The drug combination also induced the expression of mediators of the intrinsic apoptotic pathway, Bim and cleaved caspase-3, and modulated the expression of cell cycle mediators cyclin D1, cyclin B1, and phosphorylated CDK1.

The effect of metformin in EML4-ALK+ lung cancer alone and in combination with crizotinib in cell and rodent models.

Cell based studies have suggested that the diabetes drug metformin may combine with the anaplastic lymphoma kinase receptor (ALK) inhibitor crizotinib to increase ALK positive lung cancer cell killing and overcome crizotinib resistance. We therefore tested metformin alone and in combination with crizotinib in vivo, by employing a xenograft mouse model of ALK positive lung cancer. We found that 14 days of daily oral metformin (100 mg/kg) alone had a moderate but statistically significant effect on tumour growth suppression, but in combination with crizotinib, produced no greater tumour suppression than crizotinib (25 mg/kg) alone. We also reassessed the effect of metformin on EML4-ALK positive lung cancer (H3122) cell viability. Although metformin alone did have a moderate effect on cell viability (30% suppression) this was only at a clinically irrelevant concentration (5 mM) and there was no additive effect with cytotoxic concentrations of crizotinib. Moreover, metformin did not overcome crizotinib resistance in our resistant cells. Nevertheless, we were able to show that metformin induces a G1-cell cycle arrest and apoptosis alone and in combination with crizotinib. Also, consistent with earlier work, the addition of insulin-like growth factor-1 (IGF-1) to EML4-ALK positive cancer cells reduced cell killing by crizotinib. We therefore hypothesised that the effect of metformin in vivo was not due to direct cytotoxicity on cancer cells, but by modulation of IGF-1 expression. We therefore measured levels of IGF-1 in plasma taken from mice treated with metformin, but found no difference between the drug treatment and control groups. We further hypothesised that the effect of metformin could be due to modulation of thrombospondin 1 (TSP-1), which metformin has been proposed to regulatein vivo, but again we found no difference between the experimental groups. Finally, we investigated the potential for liver and kidney toxicity, as well as CYP3A based interactions, from the combination of metformin with crizotinib.

Inhibition of Mitogen-Activated Protein Kinase Kinase Alone and in Combination with Anaplastic Lymphoma Kinase (ALK) Inhibition Suppresses Tumor Growth in a Mouse Model of ALK-Positive Lung Cancer.

Anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancer most commonly arises through EML4 (Echinoderm Microtuble Like 4)-ALK chromosomal fusion. We have previously demonstrated that combination of the ALK inhibitor crizotinib with the MEK inhibitor selumetinib was highly effective at reducing cell viability of ALK-positive non-small-cell lung cancer (H3122) cells. In this study, we further investigated the efficacy of crizotinib and selumetinib combination therapy in an in vivo xenograft model of ALK-positive lung cancer. Crizotinib decreased tumor volume by 52% compared with control, and the drug combination reduced tumor growth compared with crizotinib. In addition, MEK inhibition alone reduced tumor growth by 59% compared with control. Crizotinib and selumetinib alone and in combination were nontoxic at the dose of 25 mg/kg, with values for ALT (<80 U/l) and creatinine (<2 mg/dl) within the normal range. Our results support the combined use of crizotinib with selumetinib in ALK-positive lung cancer but raise the possibility that a sufficient dose of an MEK inhibitor alone may be as effective as adding an MEK inhibitor to an ALK inhibitor. SIGNIFICANCE STATEMENT: This study contains in vivo evidence supporting the use of combination MEK inhibitors in ALK+ lung cancer research, both singularly and in combination with ALK inhibitors. Contrary to previously published reports, our results suggest that it is possible to gain much of the benefit from combination treatment with an MEK inhibitor alone, at a tolerable dose.