Genomic Analyses Shed Light on Mechanisms of Resistance to Noncovalent BTK Inhibitors in Relapsed/Refractory CLL

Although the development of noncovalent BTK inhibitors such as pirtobrutinib (LOXO-305) represents an encouraging therapeutic advancement for patients with chronic lymphocytic leukemia (CLL) and other B-cell malignancies, analyses have revealed a series of genetic mechanisms for acquired resistance to this new class of agents, according to Justin W. Taylor, MD.¹

To better understand the mechanisms of resistance to these newer-generation agents, Taylor and colleagues conducted mutational analyses in which they examined pre- and post-treatment specimens collected from a subset of 55 patients with relapsed or refractory CLL who participated on the phase 1/2 BRUIN trial (NCT03740529). Data revealed 9 patients who had acquired mechanisms of genetic resistance to pirtobrutinib.

With older covalent BTK inhibitors, patients typically developed resistance in BTK at residue C481, particularly C481S, which is the binding site of those inhibitors, Taylor noted. However, in this genomic analysis, 7 patients had acquired non-C481 BTK mutations, including V416L, A428D, M437R, T474I, and L528W; these mutations conferred resistance to both noncovalent and certain covalent BTK inhibitors. Moreover, mutations in PLCγ2 were found in all 9 patients. Further research is required to further explore these mutations and how they could impact treatment decisions for patients, Taylor added.

“The exact mutation in BTK dictates which drug [the patient] may be resistant to,” Taylor said. “For a long time, the only BTK mutations that we had to worry about were the C481 mutations. Now, however, we [have found] other mutations, and each of them is slightly different. Just be aware that [these mutations] might show up on reports, and that they may be of clinical significance.”

In an interview with OncLive^®, Taylor, assistant professor, lab head/PI, Sylvester Comprehensive Cancer Center, University of Miami Health System, discussed the significance of BTK inhibition in the treatment of patients with CLL, the genomic analyses conducted in those enrolled to BRUIN who received pirtobrutinib, and the significance of the data yielded from this research.

OncLive^®: Could you provide background on how the emergence of BTK inhibitors have transformed treatment for cancers like CLL?

Taylor: BTK inhibitors were approved in 2013 and 2014, [which means it has been] almost a decade [of us] having BTK inhibitors [that we can use in the treatment of] many diseases, but particularly for CLL, which was the focus of our study. BTK inhibitors have transformed the treatment of that [disease] because before [these agents], we were using chemotherapy for patients.

Chemotherapy may still be used in certain cases, but most patients are able to take these pills that can keep their CLL at bay. [BTK inhibitors have] transformed the way we thought of trying to completely get rid of the disease. In some patients, [BTK inhibitors] can cause complete remissions; [however,] typically, [these agents] cause partial remissions, patients are relieved of the symptoms [of CLL], and they can continue on with their regular lives.

What was the inspiration for your analyses? What did you set out to do with this research?

With any of our targeted therapies, we are learning that they can be quite effective, but we do expect that patients may develop resistance over time. That is what we saw with this first generation of BTK inhibitors. [These agents] are very effective, but patients were developing resistance at a particular mutation in BTK called C481, typically a C481S mutation. Newer drugs were being developed that may also have fewer [adverse] effects, but would also work against this C481S resistance mutation for the first-generation BTK inhibitors.

We are now using the next-generation BTK inhibitors, but [given our knowledge] about targeted therapies, we might expect that there could be some resistance. [As such, for our research, we] followed the patients, and in those who [developed] resistance to this new therapy, [we tried] to figure out how that [resistance] developed.

To perform your analysis, your team leveraged peripheral blood specimens collected from those participating on the phase 1/2 BRUIN study. Could you explain the rationale for using this specific trial for your analysis?

The first-in-human [aspect] is the phase 1 [portion of the research], and then it will then transition into phase 2 after [investigators] find a safe dose from the phase 1. [The trial is evaluating] pirtobrutinib, which is what [our team] studied. This is a noncovalent BTK inhibitor, [making it] 1 of those newer-generation agents that can also handle the C481S resistance. [The agent] also works against BTK wild-type or non-mutant BTK [disease].

There are other noncovalent BTK inhibitors, but we focused on the BRUIN study because it is 1 of the largest studies [examining] this class of drugs, and the furthest advanced in terms of the phase of the study. [The trial] has now finished [the] phase 1 [portion] and [has] entered [into] phase 2.

What should be known about the population of patients examined, and what were the methods used to analyze and validate resistance mechanisms in them?

These patients were all heavily pretreated, meaning that most had received a prior BTK inhibitor and also another class of drugs, BCL-2 inhibitors or venetoclax [Venclexta] or both, or other chemotherapies. In those patients, we noticed resistance. The drug was still very effective, even in these heavily pretreated patients, but 9 patients developed resistance.

We performed sequencing on those [9] patients and identified new BTK mutations that were not C481S; they were brand new mutations that had not been seen before with other BTK inhibitors. To validate those [mutations], we used a combination of methods. One was molecular biology, [where] we took cell lines, made them express the mutations, and then tested whether that caused resistance.

We also used structural biology, where we looked at where these mutations were occurring on BTK. [When we did that, we observed that the mutations] were all occurring in close proximity to each other at the place where the drug binds to the molecule. We also used biochemical enzymatic assays and drug-binding assays to validate that these resistance mutations, indeed, do cause resistance to these drugs.

Could you expand on the key findings from your analysis?

Although hundreds of patients were treated on this trial, we looked at a cohort of 55 patients that we treated. [Of these patients], only 9 [had] resistance identified so far. The majority of patients are still responding to the drug. This early look at resistance could give us a signal of what is to come, although we need to do further studies with more patients.

Out of those 9 patients, 7 of them had BTK mutations that were not C481S. The most common was L528W and T474I, and there were some others, as well. These [mutations] clustered within the drug-binding pocket, and they caused the drug not to be able to bind. In the other 2 patients, we identified mutations in PLCγ2, which is the downstream substrate of BTK; these were activating mutations that allowed the cells to continue signaling past BTK.

One of the surprising findings [with regard to] the BTK resistance mutations, is that 1 of the most common ones, L528W, caused BTK enzyme activity to decrease, which was unexpected. However, we were able to show that despite that, these cells continued to signal through the B-cell receptor pathway. As such, [the cells] are somehow bypassing BTK and still signaling to PLCγ2 and downstream.

Is there anything else that you wanted to add about the work done to better understand the heterogeneity of CLL cells in response to BTK inhibition?

When we started this project, we were not sure what we were going to find—whether the resistance mechanisms might be genetic or epigenetic, other mechanisms, or immune escape. We were looking very broadly.

We did find genetic mechanisms with the BTK and PLCγ2 mutations, but [in those studies,] we did some single-cell RNA sequencing. Even though the patients developed BTK mutations, we were able to find subsets of cells that were existent in the patient samples before they developed the BTK mutations, and they were not in those who did not develop resistance. There may be some preexisting, nongenetic factors that are predisposing these patients to develop resistance.

It makes sense [considering] the patients had previously been treated with other BTK inhibitors and other therapies. They had already been prone to developing resistance once, and a subset of these patients then were able to develop new BTK mutations. They may have some genetic instability or some other predisposing properties, which we would like to dig further into.

It appears that pirtobrutinib is a strong option for patients who develop resistance to prior generations of BTK inhibitors. What is being done for those in whom the agent is not as effective or who develop resistance to this newer-generation agent?

That is an important question. [As we said,] BRUIN is a phase 1/2 trial. Many of the patients that we studied were on the phase 1 portion, where they were getting just the pirtobrutinibby itself. In the phase 2 portion, now that they found a safe dose of the [agent], they are looking at combinations [that are pairing it] with venetoclax and other treatments.

As such, for patients who may have responded to pirtobrutinib but are now resistant, the idea is to add in another agent in combination with pirtobrutinibto see whether that can recover the sensitivity. We know that combinations tend to work better than single drugs alone, so maybe patients getting combination therapy will not develop resistance at all. The hope is that we can prevent this resistance from occurring, but that is still part of this clinical study.

Are there any other areas of research that you want to see further explored?

Some things that we are working on in the laboratory is [learning more about] the exact mechanisms of these mutations. We know that they prevent [BTK inhibitors] from binding, but they also decrease BTK activity. We are looking for those mechanisms [to understand] how they could bypass BTK to continue to survive and signal through the B-cell receptor.

We also want to perform more of the single-cell sequencing and look for non-BTK causes of resistance and what that predisposition to developing resistance might be. In that case, we may be able to prevent it earlier.

Finally, on the clinical side, as more patients are treated on these trials, we want to see how frequently these BTK mutations might [occur], [including] PLCγ2, and how common resistance is with the single drug. Hopefully, with the combination therapies, we do not see [this kind of resistance] happening.

The drug is being [explored] in earlier lines. [This study focused on patients with] relapsed/refractory CLL who had received prior treatment, so they may be predisposed to getting resistance. [Therefore, pirtobrutinib is being] studied in treatment-naive patients in a randomized [phase 3] study [NCT05023980] that should be opening soon. There are many studies further exploring this drug, and we have a lot more to learn.

What is your take-home message to your colleagues?

I do not want people to take home that these resistance mutations mean that [pirtobrutinib] is not going to be effective, because this [analysis] was [done in just] a subset of patients. The drug is still likely going to be very effective, just as ibrutinib [Imbruvica] has been very effective, even though we see resistance mutations [with that agent, too]. However, [we do need] to be aware of [this], and now that we are doing more genetic sequencing of tumor samples, these might be seen in reports on patients.

Reference

1. Wang E, Mi X, Thompson MC, et al. Mechanisms of resistance to noncovalent Bruton's tyrosine kinase inhibitors. N Engl J Med. 2022;386(8):735-743. doi:10.1056/NEJMoa2114110