Tyrosine kinase inhibitors (TKIs)

Inhibitors of the VEGF pathway, including the VEGF antibody bevacizumab (BV) and multitargeted VEGFR tyrosine kinase inhibitors such as vandetanib, have in some cases improved progression-free survival (PFS) and/or overall survival (OS) in patients with advanced NSCLC. While benefits in the overall NSCLC population have thus far been modest, it is clear that a subset of patients have dramatic benefit, while others may experience no benefit or even life-threatening toxicities. Furthermore, virtually all patients who respond inevitably develop therapeutic resistance. Thus, the development of markers predictive of response or resistance for VEGF inhibitors is critically needed to advance NSCLC treatment. There are currently no validated predictive markers for VEGF inhibitors, and the development of such markers is made more challenging by the impact of both tumor- and host-derived circulating factors on tumor angiogenesis. Useful predictive markers will likely need to reflect factors from both sources. We hypothesize that by comprehensively profiling angiogenesis-related CAFs and other peptides in the blood, as well as germline variations in angiogenesis-related genes, non-invasive predictive markers for identifying which patients benefit from VEGF inhibitors (alone or with chemotherapy) can be developed and validated.

The investigators of this proposal have a longstanding collaboration to develop predictive markers for VEGF inhibitors and, along with other investigators, have identified three promising and potentially complementary approaches using blood-based markers that will be tested in this grant. We have also played leadership roles in major randomized clinical trials of VEGF inhibitors, which have enabled us to leverage a unique set of resources to address these questions.

In Aim 1, we will use multiplexed CAF profiling to identify high-priority candidate predictive markers for differential benefit from VEGF inhibitors with chemotherapy compared to chemotherapy alone using two independent sets of samples from randomized phase II trials: one testing bevacizumab (BV) with chemotherapy (pemetrexed/carboplatin or Pem/C) vs. Pem/C alone (BATTLE-FL) trial, and another testing the VEGFR/EGFR tyrosine kinase inhibitor vandetanib (VAN) plus carboplatin/paclitaxel (VCP) vs. CP, in first-line NSCLC trial. We will also test CAF markers of benefit for VAN vs. standard therapy with erlotinib using specimens from a recently completed randomized phase III trial (ZEST).

In Aim 2, we will use MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectroscopy to develop a serum proteomic classifier for benefit for VEGF inhibitors, using samples from the same trials as in Aim 1. We previously used a similar approach to develop a serum proteomic marker of benefit (Veristrat) for EGFR inhibitors.

Finally, in Aim 3, we will validate a signature of SNPs from three angiogenesis-related genes that were found to be predictive of benefit for BV with CP (BCP) vs. CP in the ECOG4599 trial. This will be tested in the same three trials of VEGF inhibitors. It is worth noting that our interest is not in developing markers for a particular drug, but rather to identify markers and pathways relevant to predicting response or resistance to inhibitors of the VEGF pathway, a validated therapeutic target in NSCLC. Our data thus far suggest there will be overlap in markers for BV and VEGFR TKIs but it will be important to determine whether this is true, particularly as more potent and specific inhibitors are tested.

Impact. This project has the potential to address a critical unmet need in NSCLC by producing validated, non-invasive blood-based predictive markers for identifying patients more likely to benefit from VEGF inhibitors, and can provide critical insights into mechanisms of resistance to guide future combination regimens.

The ALK tyrosine kinase inhibitor (TKI) crizotinib has demonstrated significant activity in patients with ALK+ lung cancer, but its efficacy is limited by the development of acquired resistance. One potential resistance mechanism involves activation of the EGFR and HER2 kinases. This activation occurs in the absence of EGFR/HER2 mutation or amplification, suggesting an alternative mechanism for up-regulation of these kinases. We discovered a novel association between ALK and the EGFR/HER2 negative regulator, MIG-6. MIG-6 is known to inhibit EGFR/HER2 kinase activity through direct binding. However, to date, there is no known link between ALK and MIG-6. In our preliminary data, we demonstrate that MIG-6 protein levels decrease after ALK TKI treatment, a process abrogated by addition of a proteasome inhibitor. Furthermore, MIG-6 transcript and total protein levels are decreased in ALK+/TKI resistant compared to isogenic ALK+/TKI sensitive cells. The decline in MIG-6 correlates with an increase in EGFR and HER2 protein levels in the resistant cells. Based upon these data, we hypothesize that ALK serves as an upstream regulator of MIG-6, and that MIG-6 suppression drives EGFR/HER2 activation in the ALK TKI resistant state. To test this hypothesis, we propose to: 1) elucidate the molecular mechanisms whereby ALK regulates MIG-6 expression, and 2) determine the mechanisms whereby MIG-6 contributes to EGFR/HER2 pathway activation in TKI-resistant ALK+ lung cancer cells. The proposed studies may lead to strategies that augment or restore expression of MIG-6 and therefore will represent a novel therapeutic approach for patients with acquired resistance to ALK inhibitor therapy.

Cancer therapy needs to be better personalized to individual patients, each of whom carries a unique spectrum of mutations, inherited and/or acquired. One of the best cases for this is lung cancer, where molecular subgrouping patients and targeting their mutations has shown promise. While many groups have searched fervently for additional tumor-acquired mutations that are associated with drug sensitivity and resistance, few have emerged since the discovery of the initial mutations, such as EGFR. In contrast, we have applied our expertise in microRNAs (miRNAs), their biologic function and interaction with oncogenes, and as a result have discovered a new class of inherited, germ-line mutations that appear to have strong potential as companion diagnostics. These mutations are in a relatively unexplored region of the genome, the 3’ untranslated regions (UTRs), once thought to be junk DNA. The first such discovered biomarker, the “KRAS-variant,” is an inherited 3’UTR mutation in KRAS, which affects microRNA (miRNA) binding and KRAS pathway expression, as well as tumor biology. The KRAS-variant was first shown to be a genetic marker for an increased risk of developing non-small cell lung cancer (NSCLC). In addition, our data indicate that this variant represents an inherited form of an activated KRAS allele, one of the most important human oncogenes. Several groups have found that the KRAS-variant acts as a biomarker of poor outcomes across multiple cancers, including head and neck cancer, ovarian cancer, and colon cancer. We as well as others have shown that this biomarker is predictive of response to specific cancer treatments, and is not just prognostic of aggressive tumor biology. For example, the KRAS-variant predicts resistance to cisplatin in ovarian and head and neck cancer, resistance to cetuximab in combination with irinotecan chemotherapy, but sensitivity to cetuximab when delivered alone. Furthermore, our preliminary data in this proposal indicate that the KRAS-variant is a powerful companion diagnostic predicting poor response to specific agents, and excellent response to others. These findings, combined with evidence that this allele of KRAS can be targeted using siRNA and miRNA strategies that we have pioneered, leads us to believe that this KRAS-3’UTR mutation is a powerful companion diagnostic in lung cancer, and ultimately may be a critical new target for cancer therapy.

The genotype-directed use of EGFR TKIs in EGFR-mutant lung cancer patients has fundamentally changed the face of the disease with improved response to therapy, preserved quality of life, and prolonged progression-free survival. However, tumors that initially respond become resistant after ~ 1 year (i.e., acquired resistance, AR). The biology of AR is incompletely understood and is largely inferred from pre-clinical models. Our group at Mass General implemented a wide-scale program to perform repeat biopsies on EGFR-mutant patients with AR to first-generation EGFR inhibitors (gefitinib, erlotinib). This translational research paradigm has been instrumental in gaining insight into AR and has led to novel observations such as PIK3CA and BRAF mutations as mechanisms of AR and the abundant frequency of phenotypic changes at the time of AR like small cell lung cancer and epithelial-to-mesenchymal transitions. The repeat biopsy results have directly helped our patients as well, by informing choices about the most relevant clinical trials or alternative treatments. Currently, a new cohort of EGFR TKIs is entering the clinic: 2^nd-generation irreversible pan-HER inhibitors and 3^rd-generation T790M-specific inhibitors. Little is known about how AR develops to these newer therapies or how the biology of EGFR-mutant cancers evolves over time after exposure to sequential EGFR blockers.

Our proposal aims to discover the mechanisms of AR to next-generation EGFR TKIs and to understand the evolution of EGFR-mutant cancers as they face the selective pressure of sequential types of EGFR-directed therapies. To accomplish this we will collaborate across institutions to amass a large number of pre-treatment and post-treatment paired patient biopsies which we will interrogate via allele-specific genotyping as well as whole genome sequencing to define mechanisms of AR. In addition we will utilize an innovative technique of taking tumor tissue directly from a biopsy and deriving patient-specific in vitro and in vivo models in the laboratory. Expansion of these models will yield a larger supply of AR cancer material to aid descriptive and functional research about the biology of AR. In addition, establishment of pre-treatment patient-specific models will allow us to verify whether the mechanism of AR derived in the lab through ex-vivo therapy mirrors the eventual clinical mode of AR.