Attacking Acquired Mutations: Researchers Aim to Make Molecularly Targeted Cancer Therapies "Smarter"

August 8, 2014
Jane de Lartigue, PhD

OncologyLive, July 2014, Volume 15, Issue 7

Hailed as "new ammunition in the war against cancer" and featured in TIME magazine at the turn of the new millennium, molecularly targeted therapies have gone on to revolutionize cancer treatment. Clinical responses, however, are all too often short-lived as cancer cells become resistant.

Hailed as “new ammunition in the war against cancer” and featured in TIME magazine at the turn of the new millennium, molecularly targeted therapies have gone on to revolutionize cancer treatment. Clinical responses, however, are all too often short-lived as cancer cells become resistant.

In response to this significant challenge, researchers are attempting to understand the molecular mechanisms underlying resistance. As a result, a number of “smarter” therapies are now available that are specifically designed to treat resistant tumors and novel technologies for earlier detection of resistance have begun to emerge.

Acquired Resistance Poses Substantial Challenge

Since the early 2000s, therapies designed to interfere with specific molecules that drive cancer cell growth and proliferation have been repeatedly clinically validated in the treatment of various forms of cancer. Drugs targeting the tyrosine kinase receptors that play a central role in the cancer signaling pathways that orchestrate these cellular processes have proved particularly successful.

Yet despite these advances, the 5-year survival of many patients with cancer remains poor. As with traditional anticancer therapies, a significant limitation to the success of these “smart” drugs has proved to be the rapid acquisition of resistance by cancer cells.

Resistance to targeted therapy is typically classed as either intrinsic, in which a subpopulation of cancer cells are already resistant prior to treatment, or acquired, whereby cancer cells develop mechanisms of resistance in response to drug therapy.

If targeted therapies are to achieve their full potential, the challenge of resistance must be addressed. Fortunately, although overcoming resistance to traditional therapies has been particularly challenging, the mechanism of action of targeted therapies has become better understood, which makes understanding how resistance is acquired a simpler task. The experience in non—small cell lung cancer (NSCLC) highlights the evolving understanding of the genomic drivers of the disease and resistance pathways (Figure).

Unraveling Mechanisms of Resistance

Mechanisms of resistance are being elucidated through cancer cell line models and tumor biopsy samples. Both methods compare paired samples of pretreatment, drug-sensitive cell lines/tumor samples, with posttreatment, drug-resistant ones. Thus far, the predominant mechanisms of resistance fall into two general classes: those affecting the drug target itself, which typically are secondary genetic alterations including mutation and gene amplification; and those that are independent of the drug target, which include upregulation of alternative signaling pathways that bypass the targeted pathway. Nongenetic mechanisms of resistance have also been identified, such as epigenetic modifications and changes in drug metabolism. Studies of resistance in NSCLC have also shown that the tumor may undergo changes in its histology during drug treatment that can lead to resistance.

Ultimately, these mechanisms of resistance achieve a common goal of allowing the cancer cell to maintain its intracellular growth and proliferation signaling in spite of the presence of targeted therapeutics designed to inhibit it.

Secondary Mutations: A Common Pathway to Resistance

The most commonly observed mechanisms of acquired resistance to targeted therapies involve the development of secondary genetic mutations in the drug target.

Gateway Mutations

Many tyrosine kinase inhibitors (TKIs) were designed to bind to a conserved threonine residue on their target molecules. This threonine controls access to a hydrophobic pocket within the active site of the enzyme and has therefore been dubbed a gatekeeper amino acid. A single point mutation that changes this threonine to a bulkier amino acid disrupts the TKI’s ability to bind to its target.

This kind of mutation has been shown to be a highly conserved mechanism through which cancer cells develop resistance to targeted therapy. It was first observed in patients with NSCLC treated with inhibitors targeting the epidermal growth factor receptor (EGFR), such as erlotinib (Tarceva) and gefitinib (Iressa). The T790M gatekeeper mutation is present in 50% of patients who develop resistance to EGFR-targeted therapies, but is rarely found in patients with untreated tumors.

Analogous gatekeeper mutations have also been identified in the ALK gene (L1196M), which confers resistance to crizotinib (Xalkori) in patients with ALK-positive NSCLC, and in the KIT gene (T670I), which drives resistance to imatinib (Gleevec) in patients with gastrointestinal stromal tumors (GIST). Gatekeeper mutations have also been observed in the BCR-ABL fusion gene (T315I), leading to resistance to imatinib and other TKIs in patients with chronic myelogenous leukemia (CML).

Other Resistance-Driving Mutations

Many other mutations have been identified that drive resistance to targeted therapies, in both the target kinase and other proteins involved in downstream or alternative signaling pathways. The former typically impact the ability of the inhibitor to bind to its target or reinstates catalytic activity of the target despite the presence of the inhibitor, while the latter allow the target kinase to be bypassed.

Secondary mutations in the kinase domain of ALK have been identified in approximately 30% of patients with crizotinib-resistant tumors. In addition to the gatekeeper residue, the insertion of a threonine at amino acid 1151 (1151Tins) is a particularly common mutation. Both of these ALK mutations confer a high degree of crizotinib resistance compared with other mutations that have been identified.

More than 50 secondary mutations in the ABL kinase domain have been identified to date in addition to the T315I gatekeeper mutation. One other prominent example is the F317I mutation, which confers resistance to both imatinib and dasatinib (Sprycel) in CML.

The frequency of secondary mutations in the KIT gene is determined by the location of the primary cancer-driving mutation. Where primary mutations are located in exon 11, the cancer cell is more likely to develop secondary mutations that drive resistance to imatinib, compared with mutations in exon 9. This is believed to occur because tumors with exon 11 mutations have become more dependent on KIT signaling.

Finding Ways to Outsmart Cancer

Once mechanisms of resistance have been identified using preclinical modeling and then confirmed in patient biopsies, researchers are able to focus on strategies to overcome them. There is a growing list of FDA-approved cancer therapeutics designed specifically to treat resistant patient populations. CML was the first human cancer successfully treated by targeted therapy and is now serving as an important model for resistance; next-generation drugs to address resistance are furthest along in development.

Next-Generation BCR-ABL Inhibitors Imatinib was the original BCR-ABL inhibitor, approved for the treatment of CML in 2001. Since then, with the emergence of resistant tumors, several newer agents have been approved. These include dasatinib, nilotinib (Tasigna), bosutinib (Bosulif), and ponatinib (Iclusig). Both nilotinib and bosutinib are FDA-approved for the treatment of resistant BCR-ABL—positive disease; however, neither is effective in patients with the T315I gatekeeper mutation.

Ponatinib was designed specifically to treat this patient population and was granted accelerated approval by the FDA in late 2012 following positive results from the PACE trial, in which 449 patients with disease that was resistant or intolerant to prior TKI therapy achieved a major cytogenetic response (MCyR) rate of 54%, with a rate of 70% among patients with the T315I mutation. Data from a phase I dose-escalation study indicated that 10 of 11 patients with T315I-mutant chronic phase CML who stayed on ponatinib (an additional patient discontinued therapy) achieved MCyR, according to findings presented at the 2014 American Society of Clinical Oncology (ASCO) Annual Meeting in June.

Sales of ponatinib were temporarily suspended in 2013 in response to reports of life-threatening blood clots and severe narrowing of blood vessels, but the suspension has since been lifted with the development of revised prescribing information that includes a new black box warning, and implementation of a risk evaluation and mitigation strategy.

Next-Generation EGFR Inhibitors

Initially, irreversible or covalent EGFR inhibitors were designed to overcome resistance to EGFR TKIs. The predominant mechanism by which the T790M mutation drives resistance is through increased affinity of the mutant kinase for adenosine triphosphate (ATP); therefore, having an irreversible inhibitor should help to outcompete ATP for binding to the kinase. Afatinib (Gilotrif) was the first irreversible EGFR inhibitor to be approved by the FDA and others are in clinical trials. However, these agents have not proved effective against the T790M mutation.

A third generation of EGFR inhibitors is being developed to specifically address the issue of T790M-driven resistance. CO-1686 is in phase II clinical trials (NCT02147990) and AZD9291 in phase III trials (NCT02151981) in patients with EGFR T790M-mutant NSCLC that is resistant to other EGFR TKIs.

CO-1686 recently received a Breakthrough Therapy designation from the FDA, based on the results of an ongoing phase I/II trial that demonstrated an overall response rate (ORR) of 64% among 14 of 22 evaluable patients with EGFR T790M-mutant NSCLC. Meanwhile, phase I data from a study of AZD9291 was presented at the ASCO annual meeting, in which an ORR of 64% was observed among 89 patients with EGFR T790M-mutant NSCLC.

Studies of EGFR inhibitor resistance have shown that acquired resistance mutations may be lost following a TKI-free period. As a result, a number of strategies involving pulse dosing or alternate dosing with chemotherapy are being evaluated for EGFR TKI therapy.

Next-Generation ALK Inhibitors

A number of second-generation ALK inhibitors are in development for the treatment of ALK-positive NSCLC that is resistant to crizotinib. In April, the FDA approved ceritinib (Zykadia) for the treatment of patients with ALK-positive metastatic NSCLC who are resistant or intolerant to crizotinib. Approval was based on a trial of 163 patients in whom an ORR of 44% and a duration of response of 7.1 months was observed.

Ariad Pharmaceuticals recently announced updated phase I/II data for AP26113, which is a dual ALK/EGFR inhibitor. Among 57 crizotinib-resistant patients with ALK-positive NSCLC, there was an objective response rate of 69% and a median progression-free survival of more than 10 months.

Earlier Detection of Resistance

Repeat biopsies prior to and throughout the course of treatment are vital to following changes in tumor DNA over time in order to identify resistance as early as possible and prevent disease progression. However, performing biopsies can be problematic since the procedure is invasive and some tumors are inaccessible.

Novel noninvasive blood plasma-based methods are being developed to help overcome these limitations. These new technologies detect mutations in circulating, cell-free tumor DNA found in the blood supply. BEAMing technology (Beads, Emulsion, Amplification, and Magnetics) is one example. It takes a population of DNA molecules and amplifies them and transfers them onto magnetic beads, so that the beads are each coated with thousands of copies of the original DNA sequence.

The number of mutant DNA molecules in the population is then assessed by staining the beads with fluorescent probes and counting them using flow cytometry. This method was better than traditional tissue analysis at picking up secondary mutations in the KIT gene that govern resistance to imatinib and sunitinib (Sutent).

The tagged-amplicon deep sequencing (TAm- Seq) method allows identification of rare mutations in circulating DNA by amplifying entire genes from short overlapping amplicons. Using this method, researchers were able to identify mutations present in circulating DNA at allele frequencies as low as 2% with a sensitivity and specificity of greater than 97%.

These new methods for earlier, less invasive, cheaper, and higher throughput identification of resistance, combined with next-generation targeted drugs, have the potential to significantly improve outcomes for patients treated with targeted therapies and to allow these agents to reach their full potential as it was envisaged over a decade ago.

Figure. Pies Within Pies: Resistance Mechanisms in NSCLC

The pie charts at left depict the incidence of EGFR mutations and ALK rearrangements in lung adenocarcinoma, the most prevalent subtype of NSCLC, as reported recently by the Lung Cancer Mutation Consortium. The pie charts at right illustrate secondary mutations as described by Massachusetts General Hospital Cancer Center researchers.

NSCLC indicates non—small cell lung cancer; SCLC, small cell lung cancer; TKI, tyrosine kinase inhibitor


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Jane de Lartigue, PhD, is a freelance medical writer and editor based in Davis, California.

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