PDK1 Mediates NOTCH1-Mutated Head and Neck Squamous Carcinoma Vulnerability to Therapeutic PI3K/mTOR Inhibition

Abstract

Purpose: Head and neck squamous cell carcinoma (HNSCC) is driven largely by the loss of tumor suppressor genes, including NOTCH1, but lacks a biomarker-driven targeted therapy. Although the PI3K/mTOR pathway is frequently altered in HNSCC, the disease has modest clinical response rates to PI3K/mTOR inhibitors and lacks validated biomarkers of response. We tested the hypothesis that an unbiased pharmacogenomics approach to PI3K/ mTOR pathway inhibitors would identify novel, clinically relevant molecular vulnerabilities in HNSCC with loss of tumor suppressor function.Experimental Design: We assessed the degree to which responses to PI3K/mTOR inhibitors are associated with gene mutations in 59 HNSCC cell lines. Apoptosis in drug-sensitive cell lines was confirmed in vitro and in vivo. NOTCH1 pathway components and PDK1 were manipulated with drugs, gene editing, knockdown, and overexpression.Results: PI3K/mTOR inhibition caused apoptosis and decreased colony numbers in HNSCC cell lines harboring NOTCH1 loss-of-function mutations (NOTCH1 MUT ) and reduced tumor size in subcutaneous and orthotopic xenograft models. In all cell lines, NOTCH1 MUT was strongly associated with sensitivity to six PI3K/mTOR inhibitors. NOTCH1 inhibition or knockout increased NOTCH1 WT HNSCC sensitivity to PI3K/mTOR inhibition. PDK1 levels dropped following PI3K/ mTOR inhibition in NOTCH1 MUT but not NOTCH1 WT HNSCC, and PDK1 overexpression rescued apoptosis in NOTCH1 MUT cells. PDK1 and AKT inhibitors together caused apoptosis in NOTCH1 WT HNSCC but had little effect as single agents.Conclusions: Our findings suggest that NOTCH1 MUT predicts response to PI3K/mTOR inhibitors, which may lead to the first biomarker-driven targeted therapy for HNSCC, and that targeting PDK1 sensitizes NOTCH1 WT HNSCC to PI3K/ mTOR pathway inhibitors. Figure 6. AKT inhibition and PDK1 inhibition synergistically induce apoptosis in NOTCH1 WT HNSCC lines. A, NOTCH1 WT HNSCC cell lines were treated with increasing concentrations of MK2206, GSK2334470 (GSK233), or the MK2206-GSK233 combination at a fixed 1:1 ratio for 72 hours, and cell viability was measured with the CellTiter-Glo assay. The combination index (CI) values were calculated with CalcuSyn, and the fraction affected (F a ) and CI are plotted as heatmaps for all four cell lines. B, NOTCH1 WT (black text) and NOTCH1 MUT (red text) HNSCC cells were treated with 50 nmol/L GSK2126458 (GSK212), 10 mmol/L GSK233, 10 mmol/L MK2206, or the GSK233-MK2206 combination for 48 hours. Western blot analysis showed that the combination increased cleaved PARP (Cl-PARP) and cleaved caspase 3 (Cl-Casp3) in the NOTCH1 WT lines. C, Four NOTCH1 WT HNSCC cell lines were treated with 50 nmol/L GSK212, 10 mmol/L GSK233, 10 mmol/L MK2206, or the GSK233-MK2206 combination for 48 hours. Apoptosis was measured by Annexin V/propidium iodide assay, and the percentages of apoptotic cells are expressed as the mean AE SDs of three independent experiments. Ã , P < 0.001; ÃÃ , P < 0.05, two-way analysis of variance corrected for multiple com...

Role of HSP27 in the multidrug sensitivity and resistance of colon cancer cells

“…NOTCH1 is involved in the regulation of the Akt/mTOR signaling pathway (46,47), which is widely acknowledged to promote cancer development (22)(23)(24)(25)(26)42). The present study revealed that shHSP27 decreased NOTCH1 expression, indicating that HSP27 might regulate chemoresistance in colon cancer cells via NOTCH1 and that NOTCH1 might regulate of HSP27 in the Akt/mTOR signaling pathway.…”

“…The binding of NOTCH1 to its ligands allows DNA-binding proteins to regulate the expression of NOTCH1 target genes, which are involved in the proliferation, differentiation and apoptosis of various tumor cells (22)(23)(24). Additionally, PI3K and its effectors, including Akt and mTOR, play a key role in the proliferation and survival of tumor cells (25). The NOTCH1-Akt/mTOR signaling pathway has been recognized as a potential therapeutic target for the treatment of cancer (26).…”

Whole-exome Sequencing in Penile Squamous Cell Carcinoma Uncovers Novel Prognostic Categorization and Drug Targets Similar to Head and Neck Squamous Cell Carcinoma

“…In the era of targeted therapy, a rare cancer like PSCC, could be included with other SCCs umbrella clinical trials targeting a specific common targetable alteration. The above findings, provides the potential rationale for the ongoing and future clinical trials in patients with NOTCH1 mutated HNSC using PI3K/mTOR inhibitors, to potentially include patients with PSCC along with HNSC cohorts 46 . Knowing that the aberrant expression of the essential NOTCH coactivator of the Mastermind-like (MAML) family provides an alternative mechanism to activate NOTCH signaling in human cancers.…”

MiR-126-3p inhibits apoptosis and promotes proliferation by targeting phosphatidylinositol 3-kinase regulatory subunit 2 in porcine ovarian granulosa cells

“…As shown in Figure 2, the GC apoptosis rate of the siRNA-PIK3R2 group was significantly lower than siRNA-NC group ( Figure 2B, p<0.01), and the GC proliferation rate of the siRNA-PIK3R2 group was significantly higher than siRNA-NC group ( Figure 2C, p<0.01). Furthermore, several key genes of the PI3K pathway, insulin-like growth factor 1 receptor (IGF1R) [26], insulin receptor (INSR) [27], pyruvate dehydrogenase kinase 1 (PDK1) [28] and serine/threonine kinase 1 (AKT1) [29] were selected and detected to characterize biological functions of PIK3R2. We found that the mRNA expressions of IGF1R ( Figure 2D, p<0.01), INSR ( Figure 2D, p<0.01), PDK1 ( Figure 2D, p<0.01), and AKT1 ( Figure 2D, p<0.05) in siRNA-PIK3R2 group were all significantly lower than that in siRNA-NC group.…”