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Researchers Make Progress in Characterizing Diverse Mechanisms of KRAS Inhibitor Resistance


NEW YORK – A new detailed characterization of resistance mechanisms to KRAS-mutated cancers could help inform the development of better KRAS inhibitors and new treatment combinations.

In a study published Wednesday in the New England Journal of Medicine, researchers described how patients can become resistant to KRAS G12C inhibitors in a variety of ways, including by acquiring KRAS mutations beyond the G12C variety or through an increase in KRAS G12C copies; through alterations in genes other than KRAS, including BRAF, MET, ALK, and RET, among others; and via transformation from one cancer histology to another.

"We're very happy that this work is coming out to elucidate some of the mechanisms of acquired resistance to this very promising new class of KRAS G12C inhibitors," said Mark Awad, clinical director of Dana-Farber's Thoracic Oncology Treatment Center and one of the NEJM study's lead authors. "The hope is that a deeper understanding of resistance mechanisms will lead to some informed and more rational study designs for combination therapies to both prevent and delay, as well as overcome resistance when it happens."

KRAS G12C inhibitors have attracted significant investment and interest among drugmakers. Two well-known assets in the class include sotorasib (Amgen's Lumakras), which last month received accelerated approval from the US Food and Drug Administration for metastatic, KRAS G12C-mutated non-small cell lung cancer patients, and the investigational agent adagrasib, which Mirati Therapeutics is currently evaluating in clinical trials across multiple solid tumor types.

The advancement of these and other therapies in the clinic has allowed KRAS to shed its long-held reputation as an "undruggable" target.

Despite the enthusiasm surrounding the latest sotorasib approval, response rates are low relative to other approved targeted agents in NSCLC. In the Phase II CodeBreak-100 clinical trial, which supported sotorasib's FDA approval, the objective response rate among previously treated metastatic NSCLC patients was 37 percent, and the median progression-free survival was 6.8 months. In an ongoing Phase I/II study of adagrasib for KRAS G12C-mutated cancers, the NSCLC response rate was 45 percent. When these drugs were evaluated in KRAS G12C-mutated colorectal cancers, the response rates were 7.1 percent and 17 percent for sotorasib and adagrasib, respectively.

"That's encouraging, but certainly in other molecular subtypes of lung cancer, we've seen much higher response rates and longer durations of response," Awad said, pointing to drugs targeting EGFR and ALK alterations, which have demonstrated more durable responses. Contributing to this less robust benefit is the fact that, across trials of both KRAS G12C-targeted agents, most patients eventually went on to develop acquired resistance.

Characterizing resistance mechanisms

In their study, Awad and collaborators took a two-pronged approach to home in on the different reasons why patients may develop resistance to these agents. For the first part of their study, they investigated tumor tissue and circulating tumor DNA, or ctDNA, samples from a cohort of 38 patients with KRAS G12C-mutated cancers who received adagrasib on the Mirati-sponsored KRYSTAL-1 clinical trial and responded to the agent for at least 12 weeks but then experienced disease progression. Twenty-seven of these patients had NSCLC, 10 had colorectal cancer, and one had appendiceal cancer.

The researchers performed next-generation sequencing on tumor samples from 10 patients and ctDNA results from 32 patients using local testing at Dana-Farber, as well as tests from Guardant Health and Foundation Medicine. Four patients had both tissue-based and ctDNA NGS test results. They then compared these sequencing results within the KRYSTAL-1 trial against test results on samples taken prior to adagrasib treatment.

From this analysis, Awad and colleagues found that, at the time of resistance, four patients had developed secondary KRAS mutations beyond G12C. These included KRAS Y96C, H95Q, H95R, R68S, and H95D mutations. Several patients had developed activating mutations in KRAS G12D, G12V, and G13D, as well as G12R and Q16H. In two patients, the researchers found high-level focal amplifications of KRAS G12C itself, but no other resistance mechanisms.

Mutations beyond KRAS included those in other RTK-RAS-MAPK pathways, including an NRAS mutation in one patient, and BRAF V600E mutations in two patients. Multiple patients also developed MAP2K1/MEK1 mutations, and one patient developed an EGFR mutation. They also spotted acquired oncogenic gene rearrangements in colorectal cancer patients, including CCDC6-RET and EML4-ALK; and, in one colorectal cancer patient, several rearrangements had developed, including RAF1, BRAF, and FGFR3 rearrangements. The researchers also observed acquired MET amplification in two other patients, and saw that in two of the 10 patients who had tissue-based NGS, the cancer histology had actually transformed from lung adenocarcinoma to squamous-cell carcinoma. These patients, however, did not have any genomic resistance mechanisms as far as the researchers could tell.

"We also saw multiple concurrent resistance mutations happening in one patient simultaneously, which was particularly surprising," Awad said, pointing out that the researchers noticed this phenomenon particularly among patients who had ctDNA NGS, which may have painted a broader or more heterogenous picture of resistance compared with tissue NGS based on a single-site biopsy. "We hadn't necessarily seen examples of that [simultaneous multiple resistance mechanisms] from other molecular subtypes of lung cancer." For example, he said that this has rarely been observed in studies of resistance to EGFR inhibitors or ALK inhibitors.

The second part of the study, which Awad and colleagues referred to as "deep molecular screening," involved creating cell lines each containing a KRAS G12C mutation along with another possible second mutation in the KRAS gene. Together, these cell lines represented every possible KRAS mutation. "We were able to put all these mutant KRAS alleles on a KRAS G12C backbone, to ask, 'What secondary mutations, when seen in conjunction with the KRAS G12C mutations, could cause resistance to KRAS G12C inhibitors?'" he explained.

To answer this question, they treated the cell lines with sotorasib and a compound with an identical mode of binding to adagrasib. The cell lines reflected many of the same resistance mechanisms seen in the patient cohort, and when the researchers looked closely, they noted multiple resistance mechanisms surrounding the drug binding sites in the cell lines treated with the targeted compounds. For example, a mutation would occur at the R68 position or the Y96 position to cause resistance to both adagrasib and sotorasib.

Interestingly, some mutations, specifically mutations of the H95 KRAS variety, drove resistance to adagrasib in the cohort but did not end up having the same affect with regard to sotorasib. This finding suggested to researchers that a patient who acquires one of these mutations and stops benefiting from adagrasib could, in theory, still derive benefit from sotorasib. That being said, Awad was careful to note that there isn't any clinical evidence backing this theory, and it could be complicated by the fact that multiple patients who developed these differentially sensitive H95 mutations in the patient cohort also had other resistance mutations occurring simultaneously, which could confer resistance to both drugs.

"We don't have any clinical examples of patients switching from one G12C inhibitor to another to demonstrate that [approach]," Awad said. "I don't want to overstate that finding, but it would be important to study this further."

Switching from one targeted agent to another is not altogether a new approach; for patients with ALK-positive cancers who stop responding to a given ALK-targeted agent, for example, a second ALK inhibitor with a slightly different molecular structure might be given as an additional line of treatment. "There is some precedent for studying resistance mechanisms and then trying to sequence therapies," he said.

Future applications, next steps

Beyond potential treatment sequencing approaches, Awad and colleagues hope that their paper will encourage future studies that combine KRAS G12C inhibitors with other targeted therapies. For example, there are several ongoing clinical trials of adagrasib or sotorasib combined with RTK or SH2 inhibitors, which could potentially address resistance mechanisms relating to the RTK-RAS-MAPK pathway.

The researchers also expressed hope that drugmakers would work on developing additional KRAS inhibitors with alternative modes of binding and different allele sensitivities.

Of course, the broad variety of resistance mechanisms observed in this study would make it challenging to know which approach to take for which patients. Because these resistance mechanisms are primarily acquired mutations — meaning they wouldn't show up in patients' baseline sequencing test results — personalizing treatment approaches to overcome a given recurrence mechanism would require testing patients at the time of relapse.

That said, it's possible that one or more of these resistance mechanisms could be more frequent than the others, but it will take further studies in larger patient cohorts to improve understanding of the landscape of genetic alterations and mechanisms driving KRAS G12C inhibitor resistance.

To this end, Awad is hopeful that the launch of a large-scale, decentralized correlative study called SPARK — short for "Study of Pathways of Acquired Resistance in KRAS-driven cancers" — will add to the findings detailed in the NEJM. Any patient in the country who develops resistance to a KRAS G12C inhibitor could sign up for the study and partake in SPARK remotely, Awad explained.

"We'll send them a kit, they'll have blood drawn, and have free ctDNA assessed for resistance mechanisms," he said. "We're hoping through this nationwide study, if we get good participation, that we'll be able to have a much broader, deeper understanding of resistance across tumor types and across these different drugs."