Patterns of resistance to B cell–receptor pathway antagonists in chronic lymphocytic leukemia and strategies for management

The management of chronic lymphocytic leukemia (CLL) has changed dramatically over the past 5 years with the advent of the B-cell receptor (BCR) signaling antagonists for relapsed disease, and this paradigm shift will likely continue through the next few years as these agents are investigated as frontline therapy. Before the introduction of these agents in clinical trials, patients with relapsed disease had a generally poor outcome with standard chemoimmunotherapy or antibody therapy. Stem cell transplantation was an option for young and fit patients with relapsed disease, but this was not an option for the majority of the CLL population. In 2014, however, the U.S. Food and Drug Administration (FDA) approved two novel oral kinase inhibitors: (1) the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib; and (2) the phosphoinositide-3 kinase (PI3K) inhibitor idelalisib. These agents have changed the standard of care for patients with relapsed CLL.

Although ibrutinib and idelalisib both can induce rapid and durable remissions, it has become clear that neither of these agents are a panacea. There are patients who fail to respond and those who relapse, both early and after long periods of remission. Two mechanisms that underlie resistance to ibrutinib have been identified, and progress is underway to elucidate mechanisms of resistance to idelalisib, as well. Furthermore, clinical trials designed for patients who relapse after taking kinase inhibitors are in process, and novel therapies are being developed that provide hope for patients who relapse after BCR signaling antagonists. This review will discuss mechanisms of resistance to the oral BCR signaling antagonists ibrutinib and idelalisib and potential strategies in the management of resistant disease.

Ibrutinib is a relatively selective, irreversible inhibitor of BTK. This target is attractive because, unlike other kinases in the BCR pathway, BTK does not have natural redundancy and is selective for B cells, so inhibition leads to a B cell–specific phenotype. Both in vitro and in patients, ibrutinib has been shown to potently inhibit BCR signaling, prevent lymphocyte adhesion and homing, and inhibit protective effects of the microenvironment.^1-3  This drug is FDA approved for patients with relapsed or refractory CLL and for patients with CLL with del(17p) based on multiple studies showing benefit of this agent, although limited data exist for previously untreated patients with del(17p). In the phase 1b/2 study of ibrutinib in patients with relapsed or refractory CLL, at 30 months, the overall response rate (ORR) is 90%, with a median time to response of 1.9 months and a median time to best response of 7.4 months (range of 1.4-12.2 months).⁴  ORR increases over time with both ibrutinib and idelalisib, as lymph node–dwelling lymphocytes are released into the circulation early on in therapy and then slowly decline over time. At a median follow-up of 30 months, progression-free survival (PFS) is 69% and overall survival (OS) is 79%.⁴  The phase 3 trial that led to the approval of ibrutinib compared this agent with the cluster of differentiation (CD) 20 monoclonal antibody ofatumumab. With a median follow-up of 9.4 months, ORR was superior with ibrutinib, but more importantly both PFS (median of 8.1 months for ofatumumab versus not reached for ibrutinib) and OS (12-month estimates: 81% for ofatumumab and 90% for ibrutinib) were improved significantly with ibrutinib.⁵  A phase 2 study was also conducted specifically in patients with TP53 aberrations with either treatment-naive or relapsed disease. The cumulative incidence of progression at 24 months was 9% for previously untreated and 20% for previously treated patients. Estimated OS at 24 months was 84% for previously untreated and 74% for previously treated patients.⁶ 

Idelalisib is a reversible, p110 delta isoform–specific PI3K inhibitor. Because the delta isoform is specific for B lymphocytes, this agent has selective effects on the CLL cells with relative sparing of other hematopoietic cells. It potently inhibits PI3K signaling and can block the protective effects of the CLL microenvironment in vitro.⁷  It has been FDA approved in combination with rituximab for the treatment of relapsed/refractory CLL. In a phase 1 study of the single agent, 72% of patients achieved an objective response, with a median PFS of 15.8 months. When focusing only on patients receiving a biologically effective dose, the PFS was 32 months. Median OS has not been reached, with a 36-month OS of 75%.⁸  The definitive phase 3 study of the idelalisib plus rituximab regimen studied this combination versus placebo plus rituximab in 220 patients. At the second interim analysis, the addition of idelalisib led to an ORR of 77% (versus 15% with rituximab plus placebo) and a 12 month PFS of 66%.⁹ 

Mechanisms of resistance to the novel oral kinase inhibitors have been identified in patients who have relapsed on ibrutinib and are outlined in Figure 1. From whole exome sequencing performed on the first 6 patients with relapsed CLL after ibrutinib in which peripheral blood samples were available at baseline and at relapse, 5 of 6 patients were found to have an identical mutation in the binding site of ibrutinib (C481) in which cysteine was mutated to serine.¹⁰  In one of these patients, 3 separate mutations in phospholipase Cγ2 (PLCγ2; R665W, L845F, and S707Y), the kinase immediately downstream of BTK, were found in addition to BTK C481S, and, in one patient, a PLCγ2 R665W mutation was found without BTK mutation. Functional characterization of these mutations demonstrated that BTK C481S reduces the binding affinity of ibrutinib for BTK and allows only reversible BTK inhibition rather than irreversible. Because of the relatively short half-life of ibrutinib, this results in only transient inhibition of BTK, and, in multiple patients, it has been confirmed that patients who relapse with the C481S mutation have expression of phosphorylated BTK that is not inhibited by the administration of ibrutinib.^10,11  Of the mutations identified in PLCγ2, one had been shown previously to be gain of function (S707Y),¹²  and the remaining two mutations, R665W and L845F, were both demonstrated to be potentially gain of function, allowing activation in the presence of inactive BTK.^10-13  More recently, 8 additional patients with CLL relapsed after ibrutinib were characterized genetically using targeted deep sequencing at baseline and relapse, and all were found to have mutations in BTK at C481 or in PLCγ2.¹⁴  Importantly, there was not a clear association of BTK or PLCγ2 mutations with Richter's transformation, only CLL progression. Furthermore, another group has reported 3 patients who relapsed on ibrutinib and did not identify BTK mutations in any patient, although one had a mutation in PLCγ2,¹⁵  suggesting that there likely are relapses that are not the result of mutations in the BCR pathway.

Because it does appear that most CLL progressions on ibrutinib are the result of mutations in BTK or PLCγ2, a critical question is whether these mutations are present at baseline or whether they are acquired during therapy as a result of drug-induced pressure. Computational evolutionary models suggest that resistance mutations should be present before ibrutinib administration,¹⁶  but this has not yet been proven in patients. In both reports of ibrutinib resistance attributable to BTK or PLCγ2 mutations, deep sequencing of the peripheral blood was performed at baseline, and no mutations were identified.^10-14  Furthermore, a separate group performed deep sequencing in a cohort of 613 ibrutinib-naive patients and found no mutations in BTK at C481.¹⁷  Although these data suggest that BTK C481 mutations are not found commonly in the peripheral blood, additional study will need to be performed to determine whether these mutations are present in very small clones or in niches other than the peripheral blood.

Ibrutinib resistance has also been explored in hematologic malignancies other than CLL. In mantle cell lymphoma, BTK C481S mutations have been identified in patients with late progressions, whereas early progressions and primary resistance have not been demonstrated to be associated with this mutation.¹⁸  In Waldenstrom macroglobulinemia, mutations in C-X-C motif chemokine receptor 4 have been shown in vitro and in vivo to lead to ibrutinib resistance,^19,20  but these mutations have not been described in CLL. Furthermore, ibrutinib has shown some activity in activated B cell–like subtype diffuse large B-cell lymphoma, and preclinical data suggest that the activity is attributable to nuclear factor-κB (NF-κB) blockade and limited to those tumors with chronic active BCR signaling and wild-type caspase recruitment domain 11 (CARD11).^21,22  Recurrent mutations in CARD11 have not been observed in CLL.^23,24 

Mechanisms of resistance to idelalisib have not yet been described in CLL, although work is ongoing in this area. Because idelalisib selectively targets p110δ (encoded by PIK3CD), one possible resistance mechanism would be through mutation or upregulation of either PIK3CD itself or an alternative class 1A PI3K, either PIK3CA or PIK3CB. In mantle cell lymphoma, it has been shown in vitro that a higher ratio of PIK3CA/PIK3CD mRNA in cell lines predicts resistance to selective p110δ inhibition.²⁵  In a breast cancer cell line in which PIK3CA was mutated, resistance to a PI3Kα inhibitor was mediated by upregulation of PIK3CA, so that PI3K function was maintained even with normally saturating concentrations of the inhibitor.²⁶  Other preclinical studies primarily with p110α inhibitors in breast cancer models have noted other potential resistance mechanisms outside of the drug target, including MYC amplification.²⁷  Whether these mechanisms will be relevant to CLL remains to be seen.

There is currently no standard of care therapy for patients who relapse after BCR signaling antagonists, and therefore a clinical trial is the preferred approach for all patients. Here we will discuss agents currently available in clinical trials with scientifically driven rationale for the treatment of kinase inhibitor-resistant disease.

For patients who relapse after idelalisib, there are data suggesting that ibrutinib is effective,⁴  so this would be the preferred approach for patients who do not have contraindications to ibrutinib therapy. For patients who have already progressed on ibrutinib, are intolerant to ibrutinib, or have contraindications, such as anticoagulation with warfarin, an agent with a distinct mechanism of action may be effective. For patients with intolerance or ibrutinib contraindication, a more specific BTK inhibitor may be of clinical utility.

Relapse on ibrutinib takes two distinct forms: (1) Richter's transformation, which tends to occur early in therapy; and (2) CLL progressions, which tend to occur after 1-2 years of treatment.¹⁴  Both types of progression are associated with subsequent chemoresistance and poor survival. In our experience, median survival after Richter's transformation is 3.5 months and after CLL progression is 17.6 months.^14,28  After ibrutinib discontinuation, disease tends to be rapidly progressive, so it is important to continue ibrutinib until an alternative treatment strategy can be administered. In our institution, outside of a clinical trial, we will generally continue ibrutinib and add lymphoma-directed chemoimmunotherapy.

Besides the oral kinase inhibitors, the BCL2 inhibitor venetoclax is the most exciting therapy currently in clinical trials for CLL and may play an important role in the treatment of ibrutinib-resistant disease. Like idelalisib and ibrutinib, venetoclax is orally bioavailable and works by mimicking Bcl-2 homology 3 to antagonize BCL2 and its prosurvival family members. In a phase 1 study with a median follow-up of 15 months, the ORR was 77%, with a high complete response (CR) rate of 23%. Estimated median PFS is 18 months.²⁹  In combination with rituximab, at a median follow-up of ∼7 months, the ORR was 88%, with 6 of 49 patients experiencing disease progression during this time. This drug has been shown in clinical studies to induce minimal residual disease-negative CRs, which are generally not seen with PI3K and BTK inhibitors.³⁰  There is a study underway currently to evaluate venetoclax in patients resistant to kinase inhibitor therapy.

Because the BCR pathway retains importance after relapse from ibrutinib, as evidenced by specific mutations in the BCR pathway driving relapse, targeting this pathway even after ibrutinib relapse is of interest. Potential alternative BCR-directed strategies can be found in Figure 2. Targeting the BCR in these patients can be accomplished proximal to BTK, through BTK itself, or distal to BTK. Proximal targeting through spleen tyrosine kinase (SYK) or LYN inhibition in patients with BTK or PLCγ2 mutations at first glance appears counterintuitive but may be a very rational strategy. Both BTK and PLCγ2 mutations that drive resistance to ibrutinib require stimulation of the BCR for activation, with SYK activating PLCγ2 directly in patients with PLCγ2 mutations. Both SYK and LYN inhibitors in vitro are active in the presence of BTK and PLCγ2 mutations, including primary CLL cells from ibrutinib resistant patients.¹³  Data in lymphoma suggest that upregulation of PI3K is seen commonly in ibrutinib resistance,¹⁸  which suggests that idelalisib plus rituximab combination therapy may be successful in this scenario. Furthermore, preclinical data with the PI3K p110 gamma/delta inhibitor duvelisib suggests that this agent may be effective in patients with C481S BTK mutations.³¹  Preliminary results with IPI-145 have not shown striking efficacy,³²  but patients were not necessarily stratified based on BTK mutational status and there may be differences in post-ibrutinib response among PI3K inhibitors.

Specifically in patients with BTK C481 mutations, alternative targeting of BTK may be an option to treat resistance, and BTK inhibitors that bind BTK distinct from C481 may be of clinical utility and are under active preclinical and clinical investigation.³³  Agents that affect BTK in a manner distinct from kinase inhibition are interesting, as well. One such class of agents are the heat shock protein 90 (HSP90) inhibitors. BTK is a client protein of HSP90, and in vitro, HSP90 inhibitors have been shown to overcome ibrutinib resistance in a mantle cell line driven by alternative NF-κB signaling.³⁴  Similarly, the exportin 1/chromosomal maintenance 1 inhibitor selinexor has been shown to suppress BTK gene expression and to be effective in cell lines with BTK C481S mutations, as well as an in vivo model of ibrutinib resistance.³⁵  Targeting the BCR downstream of BTK may also be of clinical utility, and agents that target protein kinase Cβ (PKCβ)³⁶  are currently under investigation and show preclinical efficacy in CLL. Similarly, drugs that target farther downstream in the BCR pathway, such as extracellular signal-regulated kinase (ERK) and AKT, are in development and have shown efficacy in other malignancies.

Although most of the efforts at this time are focused on replacing ibrutinib with another agent in the case of progression, another strategy would be to combine a new agent with ibrutinib in these patients to potentially restore ibrutinib sensitivity. One rationale for this approach is the finding that known ibrutinib-resistant clones (containing either BTK or PLCγ2 mutations) do not occupy the entire leukemic burden. Furthermore, we have seen that disease tends to escalate quickly on ibrutinib withdrawal, suggesting that, although the patient is relapsing, the drug continues to have an anti-leukemic effect.¹⁴  Theoretically, any of the previous agents could be added to ibrutinib to try to overcome resistant disease, and there are other classes of agents with scientific rationale to combine with ibrutinib that may be of interest. One broad class would be the immunomodulatory agents, including lenalidomide and the checkpoint inhibitors. The combination of ibrutinib with lenalidomide has a number of potentially advantageous properties. Both of these agents affect the CLL microenvironment and have the potential to restore normal T-cell function.¹  Ibrutinib has been shown to induce CD4 cell T-helper 1 skewing, which is predominantly a cytotoxic phenotype and may help in both anti-tumor immunity and recovery from infectious toxicity.³⁷  Lenalidomide has been shown to induce T-cell activation and results in increased sensitivity of CLL cells to apoptosis.³⁸  Therefore, the combination of the two has the potential to restore normal T-cell function and induce anti-tumor immunity. Another potential combination strategy with ibrutinib is the immune checkpoint inhibitors, which have shown great promise in a wide spectrum of solid malignancies. Ibrutinib has been shown to reduce programmed cell death protein-1 (PD-1) expression on CD8-positive T cells,³⁹  and combinations of ibrutinib and an anti-PD-1 ligand antibody are effective in a mouse model of lymphoma that is insensitive to either agent alone and solid tumor models without BTK expression.⁴⁰ 

Finally, given the currently poor prognosis for patients who relapse after ibrutinib, an alternative strategy, such as reduced intensity allogenic stem cell transplant or chimeric antigen receptor (CAR) T-cell therapy, should be considered. Transplant may be an ideal consideration in patients who have a donor and are able to enter a remission with salvage therapy, but it is limited to younger patients with preserved performance status. CAR T cells administered within the context of a clinical trial may be an option for some patients and offers the potential for long-term disease control.

At this time, because there are not mature data to suggest an optimal course of action for patients who progress on ibrutinib, these patients should all be considered for a clinical trial. Trials of venetoclax could be considered for all patients. For patients with known mutations in BTK, I would also consider a trial investigating an alternative mechanism to target BTK (either kinase or protein) because this pathway retains importance in the presence of BTK C481 mutations. For patients with known mutations in PLCγ2, I would consider trials of a SYK or LYN inhibitor or a downstream target such as a PKCβ inhibitor. Additionally, as a more long-term strategy, eligible patients should be considered for a stem cell transplant, and consideration should be given to a trial of CAR T cells if this is a possibility.

Although the results that have been seen with the kinase inhibitors have been phenomenal thus far, there remains a need for new therapies because patients who relapse after these novel agents, especially ibrutinib, are difficult to salvage. All of these patients should be considered for inclusion on a clinical trial because most will do poorly with standard therapy, and, given the low numbers of patients who have relapsed, a coordinated effort will be needed to determine the optimal therapy in a reasonable amount of time. For patients who are candidates for stem cell transplant or CAR T cells, clinical trials of novel agents for ibrutinib-resistant disease can be considered a bridge to these more definitive therapies. As more knowledge is gained regarding the mechanisms of relapse and the risk factors for relapse, these therapies may be more accurately sequenced for individual patients. Additionally, newer-generation BTK and PI3K inhibitors may decrease the relapse rate even further or potentially treat patients with resistance to these first-generation inhibitors, offering the potential for longer remissions or even cures for our patients with CLL.

Jennifer A. Woyach, The Ohio State University, Comprehensive Cancer Center, 445A Wiseman Hall, 410 W 12th Ave, Columbus, OH 43210. Phone: 614-685-5667; e-mail: jennifer.woyach@osumc.edu.