Acquired resistance

When the cancer progresses after initial treatment

T cell receptor engineering for the treatment of RET fusion-positive NSCLC

2022

RET-positive awards

Grant title (if any)

RETpositive / LUNGevity Foundation Lung Cancer Research Award

Alexandre Reuben, PhD

University of Texas MD Anderson Cancer Center

Houston

Despite advances in the development of RET inhibitors, patients with RET fusions eventually progress. Immunotherapy has been inefficient in patients harboring RET fusions. However, RET fusion proteins themselves may be immunogenic and give rise to an immune response. Dr. Reuben hypothesizes that RET fusions give rise to immunogenic antigens which can be effectively recognized and targeted by engineered T-cells. This project will identify which antigens can elicit an immune response. This information will be used to engineer customized T-cells to gain the ability to recognize those cancer cells that produce these RET fusion proteins. The ultimate goal is to offer new therapeutic alternatives by expanding the possibility of immunotherapy treatment in the overwhelming majority of NSCLC patients harboring RET fusions.

Research Summary

RET fusions are a challenging subset of non-small cell lung cancers (NSCLC). Although initial efforts with multikinase inhibitors resulted in limited success, subsequent development of selective RET inhibitors has bolstered responses for patients harboring these genomic alterations. Despite these advances, patients with RET fusions eventually progress, highlighting the need for additional therapies. Immunotherapy has been inefficient in patients harboring RET fusions. However, RET fusions themselves may be immunogenic. T cells recognize proteins at the surface of cancer cells called antigens, upon which they can specifically destroy tumors. Importantly, fusions constitute a particularly immunogenic antigen type which could be the key to clinical responses. Although T cells harbor millions of different receptors capable of recognizing different antigens, they can be genetically-engineered to recognize an antigen of interest. As such, we hypothesize that RET fusions give rise to immunogenic antigens which can be effectively targeted by engineered T cells. To address our hypothesis, we will identify antigens derived from tumors harboring RET fusions, receptors capable of killing tumors with these antigens and engineer T cells to gain this ability. Our work could be of benefit to patients progressing on selective RET inhibitors. Our goal is to generate a library of TCRs which could be used off-the-shelf to target the two dominant RET fusions (KIF5B-RET=75%; CCDC6-RET=25%) in order to offer new therapeutic alternatives to the overwhelming majority of NSCLC patients harboring RET fusions.

Technical Abstract

Fusions in the RET proto-oncogene account for ~2% of non-small cell lung cancers (NSCLC). Despite initial failures tied to the use of multi-kinase inhibitors, recent selective RET inhibitors have proven remarkably more successful. However, patients eventually recur on these therapies, highlighting the need for other therapeutic alternatives. Immunotherapies have been unsuccessful in patients harboring RET fusions, likely in part due to their low tumor mutational burden. Neoantigens are immunogenic antigens which are derived from somatic mutations, resulting in antigen-specific killing by T lymphocytes. Importantly, insertions, deletions, and gene fusions may exhibit increased immunogenicity, due to their accrued dissimilarity with unmutated transcripts and proteins, which may provide a unique opportunity to target RET fusions using T cell receptor engineering approaches. Accordingly, we hypothesize that RET fusions are immunogenic and can be effectively recognized using T cell receptor engineering approaches. To confirm this hypothesis, we propose to identify RET fusion-derived neoantigens, identify and isolate the T cells which recognize them, and engineer T cells to kill targets in vitro. Through this work, we aim to generate a library of TCRs which could be used off-the-shelf to target the two dominant RET fusions (KIF5B-RET=75%; CCDC6-RET=25%) and 10 most prevalent HLA alleles in the United States, in order to afford new therapeutic alternatives to the overwhelming majority of patients harboring RET fusions in NSCLC.

Key words

Non-small cell lung cancer (NSCLC)

RET or rearranged during transfection

Stage III

Stage IV

Targeted therapy

Read more about T cell receptor engineering for the treatment of RET fusion-positive NSCLC

MET and EGFR as biomarkers for amivantamab in overcoming RET TKI resistance

2022

RET-positive awards

Grant title (if any)

Hamoui Foundation / LUNGevity Lung Cancer Research Award

Tejas Patil, MD

University of Colorado Denver, AMC and DC

Denver

Two possible pathways that seem to be important for resistance to RET inhibitors are the EGFR and MET signaling pathways. Conventional methods of detecting EGFR or MET resistance may not identify many cases where both pathways are involved. In this study, Dr. Patil will use several different laboratory techniques to better detect and define EGFR and MET resistance. He anticipates that the EGFR and MET pathways can be blocked by a newer drug called amivantamab, which is a bi-specific antibody that specifically targets both EGFR and MET.

Research Summary

Improved understanding of the genetics of non-small cell lung cancer (NSCLC) has led to the recognition that certain mutations in NSCLC can be effectively treated with targeted therapy. Unlike chemotherapy, targeted therapy interferes with how a cancer grows and spreads by blocking a specific mutation that a cancer cell needs to survive. An important mutation in NSCLC is the RET gene rearrangement. There have been several targeted treatments (such as selpercatinib and pralsetinib) that are highly effective in controlling RET+ NSCLC. While we can achieve durable long-term control with these RET targeted therapies, ultimately resistance is inevitable. An important category of resistance is called bypass signaling. This is when the cancer activates and recruits a different pathway to grow and survive in the presence of a RET inhibitor. Two possible pathways that seem to be important for resistance are the EGFR and MET pathways. Our main hypothesis is that EGFR and MET pathways are recruited by RET+ cancer cells in the presence of a RET inhibitor as a way for the cancer to survive and escape the effects of the targeted treatment. We suspect that the conventional methods of detecting EGFR or MET resistance may not identify many cases where both pathways are involved. In this proposal, we will attempt to use several different laboratory techniques to better detect and define EGFR and MET resistance. We anticipate that the EGFR and MET pathways can be blocked by a newer drug called amivantamab, which is a bi-specific antibody that specifically targets both EGFR and MET. I wrote an investigator initiated clinical trial that has been approved and funded by Janssen. In this clinical trial, we will have a dedicated cohort for RET+ patients and combine amivantamab with their prior RET targeted therapy. We hope this study will provide insights into the nature of EGFR and MET signaling as resistance mechanisms.

Technical Abstract

Patients with RET-rearranged non-small cell lung cancer (RET+ NSCLC) demonstrate dramatic responses to RET tyrosine kinase inhibitors (TKIs). An important category of acquired resistance is through bypass signaling that circumvents the RET pathway altogether. When a dominant pathway (such as the RET fusion) is inhibited by a TKI (such as pralsetinib or selpercatinib), recruitment of bypass signaling can allow the cancer cell to sustain critical oncogenic pathways. We have generated RET cancer cell lines resistant to both pralsetinib and selpercatinib, LC-2/ad (CCDC6-RET) and CUTO42 (EML4-RET), that demonstrate sensitivity to afatinib, suggesting that EGFR activation is a possible mechanism of resistance. Similarly, MET amplification has been observed as an important bypass signal, seen in up to 15% of RET resistant cases. However, this is likely an under-estimate of the true spectrum of MET-mediated resistance. First, there is no consensus on what copy number (or gene-to-centromere ratio) defines MET amplification in the acquired resistance setting. Second, MET amplification is likely one of many potential mechanisms of MET-mediated resistance. Finally, the level of MET signaling needed for cancer cells to survive in the presence of a RET TKIs may be different than if MET was a de novo driver oncogene. Our central hypothesis is that MET and EGFR expression are present at baseline to varying degrees among patients with RET+ NSCLC. The use of RET TKIs selects for cancer clones that efficiently and effectively utilize bypass signaling through pre-existing EGFR and MET pathways where both have been described as mechanisms of resistance in oncogene-driven subtypes of lung cancer. Amivantamab (JNJ-61186372) is an EGFR-MET bispecific antibody that has shown synergistic inhibition of tumor growth when combined with EGFR TKIs such as lazertinib. In the ongoing CHRYSALIS Phase 1/2 study of amivantamab with lazertinib, an objective response rate of 29% was seen among 28 patients who had no identifiable mechanism of resistance to EGFR or MET TKIs. One interpretation is that several patients in this analysis had EGFR or MET signaling as a mechanism of resistance that were not being captured through traditional biomarker assays. We anticipate that a similar scenario will be present among patients with RET+ NSCLC progressing on RET TKIs. I have written an investigator-initiated trial (IIT) titled “A phase 1/2, open label, study of amivantamab (JNJ-61186372) among participants with advanced NSCLC harbouring ALK, ROS1, and RET gene fusions progressing on tyrosine kinase inhibitors without identifiable mechanisms of acquired resistance” that has already been approved and funded by Janssen. This trial will have a dedicated RET cohort that will allow us an opportunity to obtain samples pre and post-amivantamab. This infrastructure will allow us to readily test the relevance of MET and

Key words

Acquired resistance

Adenocarcinoma

Epidermal growth factor receptor (EGFR)

Metastatic

Non-small cell lung cancer (NSCLC)

RET or rearranged during transfection

Stage III

Stage IV

Targeted therapy

Read more about MET and EGFR as biomarkers for amivantamab in overcoming RET TKI resistance

Novel structure-based and combinatorial approaches for RET-fusion NSCLC

2022

RET-positive awards

Grant title (if any)

Hamoui Foundation / LUNGevity Lung Cancer Research Award

John Heymach, MD, PhD

The University of Texas MD Anderson Cancer Center

Houston

There is an urgent need to identify new agents or combination therapies to benefit patients whose tumors have developed resistance to current RET inhibitors. Currently, the true extent of RET-dependent (resistance mutations in the RET gene) versus RET-independent mechanisms of resistance is unknown. Dr. Heymach’s team will study mechanisms and biomarkers of RET-independent drug resistance and test different drug combinations to overcome RET inhibitor resistance.

Research Summary

RET fusions are oncogenic drivers that are heterogeneous among patients and can occur in approximately 2-3% of lung cancer cases. The treatment of RET fusions has been revolutionized by the development of RET-targeted agents (selperactinib and pralsetinib), but inevitably resistance emerges, and the treatment of resistant disease is a clinical challenge. Resistance is caused by additional mutations in the RET gene (RET-dependent) or changes in other genes/proteins that cause tumor cell growth, but bypass RET inhibition (RET-independent). New studies are urgently needed to identify new agents or combination therapies to benefit these patients. Recently, our team successfully developed a system to classify distinct mutations based on their impact on protein structure and drug sensitivity in EGFR driven lung cancer. We hypothesize that this approach could be used to match patients with RET alterations to the best drugs for each alteration. Building on our previous experience, in this proposal we aim to developing a functional “atlas” of RET fusions and establish a structure/function-based classification approach that would inform clinicians of the best treatment options for RET fusion NSCLC and resistant disease. Further, we will determine mechanisms and biomarkers of RET-independent drug resistance and rational drug combinations to overcome RET inhibitor resistance. These studies will be translated to better guide patient selection and rationale drug combinations for future clinical studies. To this end, we have built a multidisciplinary team with strong expertise in cancer genomics, molecular modeling, and a track record of rapidly translating advances from the laboratory into clinical trials.

Technical Abstract

RET fusions occur in 2-3% cases of lung cancer and there are currently two FDA-approved RET-specific inhibitors, selpercatinib and pralsetinib, for the treatment of patients with RET fusion positive lung or thyroid cancers. Therapeutic resistance eventually emerges, however, and studies have shown that RET-dependent mechanisms of resistance include acquired resistance mutations that alter drug binding. Further, RET fusions and mutations are heterogeneous and the impact of specific fusion partners and RET variants on drug sensitivity is poorly understood. In a recent publication in Nature, our group reported that for a related driver oncogene, EGFR, both primary and acquired resistance mutations can be classified into distinct structural groups that correlate with drug sensitivity and resistance. This structure-function based classification has important advantages 1) structure-based groups predicted drug response significantly better than standard exon-based approaches, 2) this classification enabled us to match drugs for more than ~99% of atypical mutations where currently less than 50% of mutations have an FDA approved drug. In addition, preliminary preclinical and clinical data suggests that two RET-independent mechanisms of resistance- signal bypass and lineage shift-may also drive resistance even in the presence of effective RET inhibition. Applying methodologies we established for elucidating EGFR inhibitor resistance, we will develop a structure-function based classification which can immediately impact clinical decisions for selecting therapies; comprehensively characterize RET-dependent and -independent mechanisms of resistance and biomarkers to assess them; and develop rational therapeutic strategies to overcome resistance which can guide patient selection for future clinical studies.

Key words

Acquired resistance

Adenocarcinoma

Metastatic

Non-small cell lung cancer (NSCLC)

RET or rearranged during transfection

Stage III

Stage IV

Targeted therapy

Read more about Novel structure-based and combinatorial approaches for RET-fusion NSCLC

Identifying non-genomic mechanisms of RET TKI resistance

2022

RET-positive awards

Grant title (if any)

Hamoui Foundation / LUNGevity Lung Cancer Research Award

Alexander Drilon, MD

Memorial Sloan Kettering Cancer Center

New York

Many RET-positive cancers become resistant to targeted therapy for reasons not clearly based on genetic changes alone. Dr. Drilon predicts that other causes of resistance include (1) chemical changes (in the “epigenome”) that turn cancer-causing genes on or off and (2) changes in how these cancers look under the microscope (“histology”) that affect cancer behavior. Because these changes affect cell states rather than mutations, this resistance is potentially reversible, defining a key opportunity to maintain, restore, and extend sensitivity to potent and specific RET inhibitors.

Research Summary

Understanding how RET-positive lung cancers become resistant to targeted therapy is crucial. Most of what we know is limited to how these cancers develop secondary mutations (like changes in RET itself) that cause resistance. This knowledge is insufficient. Many RET-positive cancers become resistant to targeted therapy for reasons not clearly based on genetic changes alone. To develop new treatments, we need to know what causes resistance in these cancers. The goal of this project is to bridge this knowledge gap. We predict that other causes of resistance include (1) chemical changes (in the “epigenome”) that turn cancer genes on or off and (2) changes in how these cancers look under the microscope (“histology”) that affect cancer behavior. We hope to characterize these changes and expand our anticancer arsenal by developing new strategies to treat patients whose RET-positive lung cancers develop resistance in these ways. Because these changes affect cell states rather than mutations, this resistance is potentially reversible, defining a key opportunity to maintain, restore, and extend sensitivity to potent and specific RET inhibitors.

Technical Abstract

On-/off-target resistance mechanisms to RET tyrosine kinase inhibition (TKI) are not identified in ~40% of patients with RET fusion-positive lung cancers. As such, focusing solely on genomic alterations (e.g. mutation/amplification) limits our ability to develop novel therapies for all patients. This project addresses the unmet need of identifying and characterizing alternate resistance mechanisms to selective RET TKI therapy via epigenetic mechanisms leading to altered programs of gene expression. As with other oncogene-TKI pairs, we hypothesize that epigenetic re-programming and lineage plasticity drives resistance to RET TKIs. Serial pre- and post-TKI tumor samples will be interrogated (DEG/GSEA analysis) via complementary methylome (EPIC) and transcriptomic (RNAseq) profiling. Pathologic review and immunohistochemical profiling for markers of squamous (p40, CK5/6), small cell (chromogranin, synaptophysin), and epithelial-mesenchymal (E/N-cadherin, vimentin) transformation will be performed. Candidate resistance pathways will be explored in engineered isogenic overexpression/knockout models and our institutional library of xenografts generated from patients treated with a selective RET TKI on a registrational program or standard of care. Genetic manipulation of targets that we and others have associated with licensing lineage plasticity (e.g. myrAKT, MYC, EZH2, and beta-catenin) in RET fusion-positive models to induce histologic transformation will be performed. These analyses will define strategies to constrain or abrogate therapeutic resistance.

Key words

Acquired resistance

Adenocarcinoma

Metastatic

Non-small cell lung cancer (NSCLC)

RET or rearranged during transfection

Stage III

Stage IV

Targeted therapy

Read more about Identifying non-genomic mechanisms of RET TKI resistance

Innate immunity as a mechanism of TKI resistance in fusion-driven NSCLC

2021

Career Development Award

This grant was funded in part by The Huff Project

Erin Schenk, MD, PhD

University of Colorado

Boulder

Fusion-driven NSCLC is a group of lung cancers that are driven by specific changes in oncogenes. These lung cancers tend to be addicted to these oncogenes. Such fusion-driven NSCLCs are treated with targeted therapies that block the effect of the oncogenes. However, the cancer inevitably comes back because the tumors become resistant. Traditionally, fusion-driven NSCLCs have not been successfully treated with immunotherapy. Dr. Schenk is testing how these cancers can be treated with immunotherapy through another immune pathway—the innate immunity pathway.

Research Summary

In patients with non-small cell lung cancer, a subset of tumors carry a genetic alteration resulting in the expression of ALK or ROS1 fusions (FD-NSCLC). These tumors frequently respond to and are controlled by tyrosine kinase inhibitor (TKI) therapy for several years. Eventually, these tumors develop TKI resistance and some of the resistance mechanisms are readily identified in the cancer cells. In a similar portion of patients, the reason for TKI resistance development is unknown. Based on our preliminary data, we propose that the tumor microenvironment (TME), specifically the monocytes and macrophages (MoMf) surrounding the cancer cells, contributes to the development of TKI resistance. However, the potential role of the TME as a mediator of TKI resistance has not been well studied in patients with FD-NSCLC. Using cell lines derived from patients, we observed FD-NSCLC cell lines were more resistant to TKI when grown in MoMf conditioned media. This resistance was associated with increased activation of a key cancer cell signaling pathway that drove the decreased sensitivity to TKI. In pre-treatment biopsy specimens, we found that the TME of patients with FD-NSCLC contained variable levels of MoMf. We believe our preliminary data suggests MoMf within the TME influence patient outcome to TKI therapy. We will test our hypothesis in clinically relevant animal models, patient derived FD-NSCLC cell lines, and patient biopsy specimens. Ultimately, we hope to identify novel MoMf-mediated mechanisms of TKI resistance as there is an urgent need to develop new, more effective therapies for these patients.

Technical Abstract

Patients with non-small cell lung cancers bearing ALK or ROS1 fusions (FD-NSCLC) often experience an initial, marked response to tyrosine kinase inhibitors (TKI). The eventual emergence of TKI resistance in FD-NSCLC can be partially attributed to cancer-cell intrinsic mechanisms, but these do not fully explain the diversity in patient outcomes. Currently, FD-NSCLC is viewed as a “cold” tumor with only a molecular target, and the role of the tumor microenvironment (TME) in these cancers has not been well studied. As patients have limited options after progression on TKIs, there is an urgent need to identify novel drivers of resistance to develop new, more effective therapies. Based on our preliminary data, we believe innate immune cells, monocytes and macrophages (MoMf), within the FD-NSCLC TME shape tumor response to TKI therapy. Using patient derived cell lines, we found marked TKI resistance in multiple FD-NSCLC cell lines when cultured in media conditioned by MoMf. MoMf-driven TKI resistance was associated sustained pERK activity in the cancer cell. In pre-treatment specimens from patients with FD-NSCLC, multispectral tissue imaging demonstrated a range in number of MoMf within the TME. Our preliminary data suggest the presence of MoMf with the FD-NSCLC TME may alter response to therapy. We propose investigating the mechanisms of MoMf-mediated TKI resistance and potential therapeutic approaches with clinically relevant syngeneic and xenograft animal models and patient-derived FD-NSCLC cell lines. Further, we will examine biopsy specimens from patients
Immunotherapy is a treatment approach that leverages the body’s own immune system to destroy cancer cells. In non-small cell lung cancer (NSCLC), the most common type of lung cancer, immunotherapy has opened the door to durable treatment that approaches cure; unfortunately, this outcome is rare, and many NSCLCs are initially or eventually become unresponsive. It was originally thought that unresponsive malignancies had adapted to prevent immune cells from locating and interacting with the tumor. Our research suggests this is not the case: the tumor can remain inflamed and responsive, but the immune response can become exhausted. Therapies that reinvigorate the immune response (including tumor infiltrating lymphocyte [TIL] therapies) may be particularly effective in this context. In fact, our preliminary studies suggest TIL therapy can work in up to a third of treatment-resistant NSCLCs. Capitalizing on these new insights, our proposal seeks to 1) determine pre-treatment features of sensitivity and resistance to TIL therapy in NSCLC and 2) characterize on-treatment patterns of immune response, focusing on the cells that identify and kill NSCLC cells. Accomplishing these aims will fill critical knowledge gaps, identifying characteristics of tumors likely to respond, or that fail to respond, to TIL treatment. These studies will define a roadmap, guiding application of subsequent TIL research for lung cancer. Taken together, these studies will provide crucial insights into novel treatment approaches for patients with few alternatives and an otherwise grim prognosis.

Key words

Acquired resistance

Adenocarcinoma

Immune checkpoint inhibitors

Immunotherapy

Non-small cell lung cancer (NSCLC)

Read more about Innate immunity as a mechanism of TKI resistance in fusion-driven NSCLC

Molecular Characterization of Lineage Plasticity

2021

EGFR Resisters / LUNGevity Research Award

Helena Yu, MD

Memorial Sloan Kettering Cancer Center

New York

As a mechanism of resistance to EGFR inhibitors, cancers can change histology from adenocarcinoma to small cell or squamous cell lung cancer. Once this happens, EGFR inhibitors are no longer effective treatment; there are no strategies currently available to prevent or reverse transformation after it has occurred. Dr. Yu will use advanced molecular techniques to identify genetic changes that contribute to transformation. Understanding these genetic changes will identify biomarkers that can be utilized to develop treatments to prevent and reverse transformation.

Research Summary

Metastatic EGFR-mutant lung cancer remains an incurable disease despite significant advances in cancer therapeutics. With better EGFR inhibitors, we have identified new mechanisms of resistance that are independent of EGFR signaling, notably small cell and squamous cell histologic transformation. Targeted therapies are ineffective and survival is shortened once transformation has occurred, and no strategies are available to prevent or reverse transformation after it has occurred.

Mutations in EGFR, TP53, and RB1 are associated with transformation, but additional genetic or epigenetic changes must occur for transformation to happen. We will use advanced molecular techniques to identify the mutations and cell signaling pathways that drive transformation in order to identify biomarkers that can be utilized to develop treatments to prevent and reverse transformation. Treatments will then be validated in our unique pre-clinical models which can be rapidly translated into clinical trials, providing treatments for these aggressive cancers.

Technical Abstract

EGFR-mutant lung cancers (LC) serve as the prototype oncogene-driven cancer and mechanisms of resistance to EGFR therapy are well-established. Selective pressure of EGFR inhibitors can induce lineage plasticity to escape oncogene-dependence. While we have shown EGFR/TP53/RB1-mutations are necessary but insufficient to induce small cell transformation, no other genomic signatures and transcriptomic/epigenomic effectors that induce transformation have been identified.

Using parallel exome sequencing, bulk RNA sequencing, and bisulfite sequencing, we will characterize the biomarkers of transformation in EGFR/TP53/RB1-mutant LC and mixed adenocarcinoma/squamous cell tumors. We will also utilize single-cell RNA sequencing to identify the transcriptional changes associated with transformation and to identify distinct cell types within a heterogenous tumor. We will reconstruct a phenotypic landscape to identify subclones in a transitional state between histologies. The information obtained from these studies will nominate novel biomarkers of lineage plasticity that will be validated and proposed for study in clinical trials.

Key words

Acquired resistance

Adenocarcinoma

Biomarker or biomarker testing

Epidermal growth factor receptor (EGFR)

Metastatic

Molecular profile or molecular testing

Mutation profile

Non-small cell lung cancer (NSCLC)

Small cell lung cancer (SCLC)

Squamous cell lung cancer

Stage III

Stage IV

Targeted therapy

Tumor microenvironment

Tyrosine kinase inhibitors (TKIs)

Read more about Molecular Characterization of Lineage Plasticity

Targeting Drug Tolerant States + DNA Damage to Block Osimertinib Resistance

2021

EGFR Resisters / LUNGevity Research Award

Christine Lovly, MD, PhD

Vanderbilt University Medical Center

Nashville

Despite high tumor response rates, patients treated with EGFR targeted therapies, such as osimertinib, inevitably develop disease progression. Mechanisms of drug resistance remain incompletely understood on both a genomic and proteomic level. The objective of Dr. Lovly’s project is to find new targeted treatments and drug combinations that can tackle cancer evolution and osimertinib resistance.

Research Summary

The problem of EGFR TKI resistance has not been solved. Despite the impressive anti-tumor results observed for some lung cancer patients treated with immune checkpoint inhibitors targeting PD-1/PD-L1, these benefits do not seem to extend to patients whose tumors harbor EGFR mutations. As a result, there is an urgent need to optimize and extend the benefit of TKI therapy.

With osimertinib’s utilization in the first-line setting, there are now no FDA-approved targeted therapy options for patients who develop osimertinib resistance. The majority of studies to date have focused on treating osimertinib resistance once the resistance has already developed and the patient has signs of tumor growth. However, our focus is centered upon the development of up-front combination regimens that will block resistance before it emerges, analogous to the use of combination anti-microbials therapies for treatment of patients with TB and HIV.

We focus on analysis of drug-tolerant persistor cells (DTPCs) – the tumor that we can still see on scans in the clinic, even after patients have had a partial response to osimertinib. We seek to take partial shrinkage to complete tumor regression using cutting edge single cell biology techniques. We have assembled a team of expert investigators working collaboratively to tackle this pressing issue and develop new treatments for our patients in a complete bench to bedside manner. We also aim to train the next generation of researchers so that there is a robust and diverse pipeline in place to continue to advance lung cancer studies.

Technical Abstract

Despite high tumor response rates, patients treated with osimertinib inevitably develop disease progression. Mechanisms of both intrinsic, adaptive, and acquired drug resistance remain incompletely understood on a genomic and proteomic level. Our overall objective is to find new targeted treatments and drug combinations that can tackle cancer evolution and drug resistance.

We seek to:

Understand biological determinants of response prior to therapy (poor prognosis features)
Utilize correct combination therapies in the first line to enhance response, based on biological features of subpopulations present at the start
Reprogram cells toward a more drug-sensitive state through understanding network plasticity.

More specifically, our aims are:

To define the genomic and proteomic characteristics of drug-tolerant persister cells (DTPCs) under dynamic treatment with osimertinib. Our goal is to identify new vulnerabilities in EGFR-mutant tumors which, when therapeutically targeted, can maximize the depth and duration of benefit to first-line osimertinib therapy.
To decipher mechanisms of altered cell cycle progression and osimertinib-induced DNA damage that drive tumor progression.

We have assembled a collaborative, multi-disciplinary team of laboratory scientists and clinical investigators combined with cutting edge technologies to assess genetic and proteomic heterogeneity at the single cell level, access to a robust pipeline of patient samples, and strong preliminary data to support our studies. The proposed aims are expected to define the molecular determinants of tumor heterogeneity and to develop rational therapeutic strategies for delaying / overcoming resistance. These synergistic aims are highly relevant to the current landscape of EGFR-mutant lung cancer.

Key words

Acquired resistance

Adenocarcinoma

Epidermal growth factor receptor (EGFR)

Metastatic

Non-small cell lung cancer (NSCLC)

Stage III

Stage IV

Targeted therapy

Read more about Targeting Drug Tolerant States + DNA Damage to Block Osimertinib Resistance

Overcoming ALK resistance with covalent cysteine-reactive inhibitors

2020

ALK Positive

A. John Iafrate, MD. PhD

Massachusetts General Hospital

Boston

Liron Bar-Peled, PhD

Massachusetts General Hospital and Harvard Medical School

Boston

Research Summary

The treatment of ALK fusion-positive lung cancer has been revolutionized by precision medicines including ALK kinase inhibitors resulting in improved survival for most patients. However, as we learned with crizotinib and with subsequent ALK inhibitors, most patients eventually relapse with treatment resistant tumors. Unfortunately, once resistance to multiple kinase inhibitors develops, there are few effective options available. Here, we propose to address this urgent need by developing novel inhibitors which target ALK-rearranged lung cancer. Using cutting-edge chemical proteomic technologies, we will develop therapeutic inhibitors that bind tightly to the ALK fusion proteins and cause the protein structure to become inactive. We will test the efficacy of these new inhibitors in reliable cell models of ALK tumors to demonstrate that they can induce tumor cell death. If successful, our program should deliver frontier inhibitors for ALK fusions, providing critical knowledge to direct the development of transformative lung cancer therapies with the hope of making ALK-fusion positive lung cancer a chronic disease.

Technical Abstract

The current standard of care for ALK rearranged non-small cell lung cancer (NSCLC) is targeted therapy with ALK tyrosine kinase inhibitors (TKIs) which bind within the kinase domain of ALK. Due to the development of ALK resistance mutations or bypass activation of parallel or downstream signal pathways, patients inevitably relapse. Even with the introduction of second or third generation TKIs, median overall survival has been estimated to be 6.8 years . Recent advances in immunotherapy have demonstrated progress in lung cancer treatment. However, a majority of ALK positive NSCLC patients have yet to realize the benefits from checkpoint inhibitors or CAR-T cell therapies due to primary resistance or toxicity. Therefore, the development of new treatments for ALK-rearranged NSCLC is an area of critical need. We believe that an effective assault on ALK-driven tumors requires the implementation of creative strategies that allow us to expand our arsenal of drugs to target ALK beyond its kinase domain. To meet this challenge, we have developed and employed cysteine druggability mapping (CDM), a method to define the landscape of inhibitor-protein interactions in a cancer cell. CDM identifies cysteines that can bind to covalent drug-like molecules which serve as early prototypes on which to base cancer drug development programs.
Cysteine-reactive covalent inhibitors have already emerged as an effective approach to targeting protein kinases. Osimertinib, a third-generation epidermal growth factor receptor (EGFR) TKI covalent inhibitor, overcomes EGFR T790M resistance mutations following treatment with EGFR TKI, demonstrating significant improvements over standard EGFR TKIs when used as first line therapy. Importantly, targeting cysteines with covalent inhibitors massively expands the number of proteins which are targetable with small molecule inhibitors, including many targets previously considered undruggable.

We will use ALK fusion positive cancer cell lines, together with tumor tissues from patients who developed resistance mechanisms, to generate druggable cysteine profiles by CDM. Druggable proteins specific to ALK fusion positive samples, including EML4-ALK itself, will be prioritized in our screening for protein degraders from a library of over 2,500 advanced cysteine reactive compounds. After screening, we will assess the specificity of the compound hits and validate their anti-tumor activity using ALK fusion positive cell lines and patient derived ALK TKI-resistant cell lines. We consider the use of the novel, cutting-edge cysteine targeting technology to screen for potent therapeutic compounds as a valuable proposition to improve management of patients with ALK positive lung cancer, with the goal of transforming this cancer into a chronic condition.

Key words

Acquired resistance

Anaplastic lymphoma kinase (ALK)

Biomarker or biomarker testing

Metastatic

Molecular profile or molecular testing

Mutation profile

Non-small cell lung cancer (NSCLC)

Stage III

Stage IV

Tyrosine kinase inhibitors (TKIs)

Read more about Overcoming ALK resistance with covalent cysteine-reactive inhibitors

Overcoming bypass signaling to enhance clinical responses in ALK-positive lung cancer

2020

ALK Positive

Ibiayi Dagogo-Jack, MD

Massachusetts General Hospital

Boston

Research Summary

Despite initial sensitivity to treatment with ALK tyrosine kinase inhibitors (TKIs), ALK-rearranged (ALK+) lung cancers invariably develop resistance. The majority of ALK+ lung cancers will eventually acquire ALK-independent survival mechanisms that allow them to escape ALK inhibition. These ALK-independent survival signals can originate from alternative growth receptors (“bypass tracks”). We have recently identified activation of MET signaling resulting from increased copies of the MET gene in approximately 20% of ALK+ tumors that are resistant to next-generation ALK TKIs. In the laboratory, combining lorlatinib with crizotinib (an ALK/MET TKI) overcame resistance caused by MET signaling. Based on these findings, we will conduct a trial to evaluate whether this promising combination can induce durable responses in ALK+ tumors with MET amplification. As many different bypass signals can mediate ALK-independent resistance in the remaining tumors, our trial will also evaluate whether dual targeting of ALK and MEK or SHP2 (proteins that serve as common signaling hubs for many different receptors) can broadly overcome resistance. During the study, serial biopsies will be performed to assess changes in gene and protein expression induced by treatment. This unique trial will also assess feasibility of using plasma findings to select patients with sensitive ALK+ NSCLC who may benefit from an early combination approach. The overarching goal of these studies is to develop multiple promising strategies for overcoming ALK-independent resistance.

Technical Abstract

With the development of increasingly potent and selective ALK tyrosine kinase inhibitors (TKIs), resistance is commonly mediated by ALK-independent mechanisms, such as bypass pathway activation. As bypass signaling dependencies vary across ALK-rearranged (ALK+) lung cancers, a multipronged approach is needed to broadly suppress potential bypass signals. We have identified MET activation in 20% of ALK+ tumors that are resistant to next-generation ALK TKIs. In preclinical studies, dual ALK/MET blockade re-sensitized MET-amplified ALK+ tumors to lorlatinib. MEK and SHP2 have recently been identified as important effector proteins downstream of several bypass signaling pathways. In ALK+ models harboring diverse bypass signals, pairing an ALK TKI with either a MEK or SHP2 inhibitor demonstrated broad anti-proliferative activity. Based on these findings, we will conduct a clinical trial to evaluate lorlatinib in combination with a MET, MEK, or SHP2 inhibitor for patients with ALK+ lung cancers with ALK-independent resistance. Serial tumor biopsies will be analyzed to characterize molecular determinants of response to combination therapy and identify expression changes induced by treatment. This innovative multi-arm trial will also assess whether dynamics of genetic alterations in plasma can be used to guide intensification of treatment from monotherapy to early combination therapy for patients who have not yet developed resistance to ALK-directed therapy. Collectively, the studies in this grant strive to develop diverse strategies for overcoming ALK-independent resistance, a heterogeneous molecular entity that undermines efficacy of current ALK therapeutics.

Key words

Acquired resistance

Anaplastic lymphoma kinase (ALK)

Biomarker or biomarker testing

Circulating tumor DNA

Metastatic

Molecular profile or molecular testing

Mutation profile

Non-small cell lung cancer (NSCLC)

Stage III

Stage IV

Tyrosine kinase inhibitors (TKIs)

Read more about Overcoming bypass signaling to enhance clinical responses in ALK-positive lung cancer

Phase 1 first in-human clinical trial with a therapeutic ALK vaccine in patients with ALK+ NSCLC

2020

ALK Positive

Mark Awad, MD, PhD

Dana-Farber Cancer Institute

Boston

Roberto Chiarle, MD

Harvard University

Cambridge

Research Summary

About 5% of non-small cell lung cancers (NSCLCs) have DNA mutations in the anaplastic lymphoma kinase (ALK) gene, and patients with this “ALK-positive” subtype of lung cancer are typically young and have never, or only lightly, smoked. For ALK-positive NSCLC, there are a number of FDA-approved oral ALK inhibitor pills, including alectinib, lorlatinib, and brigatinib. While these targeted therapies are initially very effective, the benefit of each of these drugs is usually limited to only a few years because ALK-positive lung cancers almost always develop drug resistance through a variety of complex mechanisms. Although PD-1 inhibitors such as pembrolizumab (Keytruda) have revolutionized the treatment of lung cancer in general, particularly in smoking-associated cancers, most patients with ALK-positive lung cancer do not respond to existing immunotherapies.

We developed an ALK vaccine immunotherapy, composed of small pieces of the ALK protein, which is an effective treatment in animal models of ALK-positive lung cancer. We now plan to launch a first-in-human clinical trial to test this ALK vaccine in patients whose cancer is growing despite treatment with an ALK inhibitor. The vaccine will be tested in combination with an approved ALK inhibitor or with an approved immunotherapy (pembrolizumab). We will also study immune cells in the blood and in tumor biopsies both before and after vaccination to ensure that this novel therapy is generating a proper anti-ALK immunologic response as we would expect. Our goal is to develop a safe and potent ALK vaccine to improve outcomes for our patients.

Technical Abstract

Several tyrosine kinase inhibitors (TKIs) are available for the treatment of ALK+ NSCLC, but resistance to all ALK TKIs (including alectinib, brigatinib, lorlatinib) has been reported and occurs through a variety of mechanisms. Once patients develop resistance to available ALK inhibitors, they are typically treated with cytotoxic chemotherapy rather than immunotherapy since response rates to PD-1 pathway inhibitors in this population are very low. However, our work in mouse models of ALK+ NSCLC has demonstrated that a vaccine generated against the rearranged portion of ALK can both prevent the development of ALK+ tumors and also more effectively treat them once they arise. We have also recently found evidence for anti-ALK immune responses in patients, as a subset of them generate spontaneous ALK autoantibodies.

We aim to translate our preclinical findings to a first-in-human clinical trial. Aim 1 will test the safety and efficacy of a novel ALK vaccine + an amphiphilic CpG toll-like receptor 9 agonist adjuvant, administered in combination with an ALK TKI or PD-1 inhibitor in patients with TKI-resistant ALK-positive NSCLC. In Aim 2, using liquid chromatography data-independent acquisition mass spectrometry, we will directly identify antigenic ALK peptides presented by MHC molecules on the surface of cancer cells from tumor biopsies obtained on trial participants. In Aim 3, we will study pre- and post-vaccination anti-ALK T-cell responses among trial participants by analyzing peripheral leukocytes and intratumoral immune cells. The overarching goal is to develop a vaccine that stimulates patients’ immune cells to recognize and eliminate ALK+ tumors.

Key words

Acquired resistance

Anaplastic lymphoma kinase (ALK)

Biomarker or biomarker testing

Immunotherapy

Metastatic

Non-small cell lung cancer (NSCLC)

Stage III

Stage IV

Tumor microenvironment

Tyrosine kinase inhibitors (TKIs)