Activating mutations in PIK3CA, the gene encoding phosphoinositide-(3)-kinase α (PI3Kα), are frequently found in estrogen receptor (ER)–positive breast cancer. PI3Kα inhibitors, now in late-stage clinical development, elicit a robust compensatory increase in ER-dependent transcription that limits therapeutic efficacy. We investigated the chromatin-based mechanisms leading to the activation of ER upon PI3Kα inhibition. We found that PI3Kα inhibition mediates an open chromatin state at the ER target loci in breast cancer models and clinical samples. KMT2D, a histone H3 lysine 4 methyltransferase, is required for FOXA1, PBX1, and ER recruitment and activation. AKT binds and phosphorylates KMT2D, attenuating methyltransferase activity and ER function, whereas PI3Kα inhibition enhances KMT2D activity. These findings uncover a mechanism that controls the activation of ER by the posttranslational modification of epigenetic regulators, providing a rationale for epigenetic therapy in ER-positive breast cancer.

On-target resistance to next-generation TRK inhibitors in TRK fusion–positive cancers is largely uncharacterized. In patients with these tumors, we found that TRK xDFG mutations confer resistance to type I next-generation TRK inhibitors designed to maintain potency against several kinase domain mutations. Computational modeling and biochemical assays showed that TRKAG667 and TRKCG696 xDFG substitutions reduce drug binding by generating steric hindrance. Concurrently, these mutations stabilize the inactive (DFG-out) conformations of the kinases, thus sensitizing these kinases to type II TRK inhibitors. Consistently, type II inhibitors impede the growth and TRK-mediated signaling of xDFG-mutant isogenic and patient-derived models. Collectively, these data demonstrate that adaptive conformational resistance can be abrogated by shifting kinase engagement modes. Given the prior identification of paralogous xDFG resistance mutations in other oncogene-addicted cancers, these findings provide insights into rational type II drug design by leveraging inhibitor class affinity switching to address recalcitrant resistant alterations. Significance: In TRK fusion–positive cancers, TRK xDFG substitutions represent a shared liability for type I TRK inhibitors. In contrast, they represent a potential biomarker of type II TRK inhibitor activity. As all currently available type II agents are multikinase inhibitors, rational drug design should focus on selective type II inhibitor creation. This article is highlighted in the In This Issue feature, p. 1

AXL overexpression is a common resistance mechanism to anticancer therapies, including the resistance to BYL719 (Alpelisib) — the p110α isoform specific inhibitor of phosphoinositide 3-kinase (PI3K) — in esophagus squamous cell carcinoma (ESCC) and head and neck squamous cell carcinoma (HNSCC). However, the mechanisms underlying AXL overexpression in resistance to BYL719 remain elusive. Here, we demonstrate that the AP-1 transcription factors c-JUN and c-FOS regulate AXL overexpression in HNSCC and ESCC. The expression of AXL was correlated with that of c-JUN both in HNSCC patients and in HNSCC and ESCC cell lines. Silencing of c-JUN and c-FOS expression in tumor cells downregulated AXL expression and enhanced the sensitivity of human papilloma virus–positive (HPV ^Pos ) and –negative (HPV ^Neg ) tumor cells to BYL719 in vitro. Blocking of JNK using SP600125 in combination with BYL719 showed a synergistic antiproliferative effect in vitro, which was accompanied by AXL downregulation and potent inhibition of the mTOR pathway. In vivo, the BYL719–SP600125 drug combination led to the arrest of tumor growth in cell line–derived and patient-derived xenograft models, as well as in syngeneic head and neck murine cancer models. Collectively, our data suggest that JNK inhibition, in combination with anti-PI3K therapy, is a new therapeutic strategy that should be tested in HPV ^Pos and HPV ^Neg HNSCC and ESCC patients.

Significance: Bioinformatic analysis revealed that approximately 40% of human messenger RNAs contain upstream open reading frames (uORFs) in their 5′ untranslated regions. Some of these sequences are translated, but the function of the encoded peptides remains unknown. Our study revealed a uORF encoding for a peptide exhibiting kinase inhibitory activity. This uORF, upstream of a PKC family member, possess the typical pseudosubstrate motif, which autoinhibits the catalytic activity of all PKCs. Using mouse models and human cells, we show that this peptide inhibits cancer cell survival, tumor progression, invasion, and metastasis and synergizes with chemotherapy by interfering with DNA damage response. Together, we point to a previously unrecognized function of a uORF-encoded peptide as a kinase inhibitor, pertinent to cancer therapy.

Most head and neck cancer (HNC) patients are resistant to cetuximab, an antibody against the epidermal growth factor receptor. Such therapy resistance is known to be mediated, in part, by stromal cells surrounding the tumor cells; however, the mechanisms underlying such a resistance phenotype remain unclear. To identify the mechanisms of cetuximab resistance in an unbiased manner, RNA-sequencing (RNA-seq) of HNC patient-derived xenografts (PDXs) was performed. Comparing the gene expression of HNC-PDXs before and after treatment with cetuximab indicated that the transforming growth factor-beta (TGF-beta) signaling pathway was upregulated in the stromal cells of PDXs that progressed on cetuximab treatment (Cetuximab^Prog-PDX). However, in PDXs that were extremely sensitive to cetuximab (Cetuximab^Sen-PDX), the TGF-beta pathway was downregulated in the stromal compartment. Histopathological analysis of PDXs showed that TGF-beta-activation was detected in cancer-associated fibroblasts (CAFs) of Cetuximab^Prog-PDX. These TGF-beta-activated CAFs were sufficient to limit cetuximab efficacy in vitro and in vivo. Moreover, blocking the TGF-beta pathway using the SMAD3 inhibitor, SIS3, enhanced cetuximab efficacy and prevented the progression of Cetuximab^Prog-PDX. Altogether, our findings indicate that TGF-beta-activated CAFs play a role in limiting cetuximab efficacy in HNC.

Genomic alterations (GA) in PIK3CA leads to the hyper-activation of the phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K) pathway in more than 20% of ovarian cancer (OC) patients. Therefore, PI3K therapies are under clinical evaluation for this subset of patients. Evidently, in clinical trials testing the efficacy of isoform-specific inhibitors of PI3K (PI3Ki), patients having a stable disease eventually relapse, as tumors become resistant to treatment. Hence, there is an urgent clinical need to develop new therapeutic combinations to improve the efficacy of PI3Ki in PIK3CA-driven OC patients. Here we identified the molecular mechanism that limits the efficacy of the beta-sparing PI3Ki, Taselisib (GDC0032), in PIK3CA-mutated OC cell lines (IGROV1 and OAW42) that acquired resistance to GDC0032. By comparing the molecular profile of GDC0032-sensitve and -resistant OC cell lines, we found that AKT/mTOR inhibition is required for GDC0032 efficacy. In resistant cells, the sustained activation of AKT/mTOR was regulated by the upregulation of the insulin growth factor 1 receptor (IGF1R). Knockdown of IGF1R re-sensitized cells to GDC0032 in vitro, and the combination of AEW541, an IGF1R inhibitor, with GDC0032 exhibited potent anti-tumor activity in vitro and in vivo. We further demonstrated that IGF1R regulates tumor cell proliferation in IGROV1 cells, whereas in OAW42, it determines autophagy as well. Overall, our findings suggest that the dual inhibition of PI3K and IGF1R may be considered as a new therapeutic strategy in PIK3CA-driven OC.