MYC Inhibition Potentiates CD8+ T Cells Against Multiple Myeloma and Overcomes Immunomodulatory Drug Resistance.

PURPOSE: Immunomodulatory drugs (IMiDs), such as lenalidomide and pomalidomide, are cornerstone therapies in Multiple Myeloma (MM), yet patients inevitably become refractory. IMiDs exert cytotoxicity through inducing Cereblon-dependent proteasomal degradation of IKZF1 and IKZF3, resulting in downregulation of the oncogenic transcription factors IRF4 and MYC. To date, clinical IMiD resistance independent of CRBN or IKZF1/3 has not been well-explored. Here, we investigated the roles of IRF4 and MYC in this context. EXPERIMENTAL DESIGN: Using bone marrow aspirates from patients with IMiD naïve or refractory MM, we examined IKZF1/3 protein levels and IRF4/MYC gene expression following ex vivo pomalidomide treatment via flow cytometry and qPCR. We also assessed ex vivo sensitivity to the MYC inhibitor, MYCi975, using flow cytometry. RESULTS: We discovered that while pomalidomide frequently led to IKZF1/3 degradation in MM cells, MYC gene expression was unaffected by pomalidomide in most IMiD refractory samples. We subsequently demonstrated that MYCi975 exerted strong anti-MM effects in both IMiD naïve and refractory samples. Unexpectedly, we identified CD8+ T cells from patients with MM as crucial effectors of MYCi975-induced cytotoxicity in primary MM samples, and we discovered MYCi975 enhanced the cytotoxic functions of memory CD8+ T cells. We lastly observed synergy between MYCi975 and pomalidomide in IMiD refractory samples, suggesting restoring MYC downregulation can re-sensitize refractory MM to IMiDs. CONCLUSION: Our study supports the concept that MYC represents an Achille's heel in MM across disease states and that MYCi975 may be a promising therapeutic for patients with MM, particularly in combination with IMiDs.

Ex Vivo Efficacy of SAR442257 Anti-CD38 Trispecific T-cell Engager in Multiple Myeloma Relapsed After Daratumumab and BCMA-targeted Therapies.

T cell-engaging antibodies (TCEs) are showing promising efficacy in relapsed/refractory multiple myeloma, even in patients that relapsed after B-cell maturation antigen (BCMA)-targeted therapy. Patients with multiple myeloma may have compromised T-cell health unaccounted for by preclinical models. Here, we use Myeloma Drug Sensitivity Testing (My-DST) for ex vivo measurement of anti-multiple myeloma cytotoxicity for the trispecific CD38/CD28xCD3 TCE SAR442257 through activation of the patients' own endogenous T cells to inform clinical development of the compound in multiple myeloma. My-DST incubates primary mononuclear cells in humanized media for 48 hours followed by flow cytometry for multiple myeloma cell viability with or without drug treatment. SAR442257 was tested on 34 samples from patients with multiple myeloma across disease settings. Potential biomarkers, T-cell dependence, and degranulation were assessed. SAR442257 was effective at low dose in My-DST cultures. High ex vivo response rates were observed in primary aspirates taken from patients with multiple myeloma at diagnosis, with modestly reduced response in multiple myeloma recently treated with anti-CD38 mAbs. SAR442257 was highly effective in patients relapsing after BCMA therapy. The CD38/CD28xCD3 trispecific format was substantially more effective than a conventional bispecific CD38/CD3 antibody format and CD38 mAbs. Anti-multiple myeloma cell cytotoxicity was dependent on the presence of endogenous T cells. Surface CD38 expression was the strongest biomarker of TCE response. My-DST is capable of measuring T cell-dependent killing using the multiple myeloma patient's own bone marrow-derived T cells. SAR442257 shows promise for multiple myeloma and may be best suited for patients declared resistant to both CD38 mAbs and BCMA-targeted therapy. SIGNIFICANCE: This study introduces the use of My-DST to measure and characterize sensitivity to anti-CD38 T-cell engager SAR442257 in primary samples using matched endogenous T cells. Preclinical testing in samples from patients with diverse treatment history supports further testing in post-chimeric antigen receptor T-cell multiple myeloma.

CD38 x ICAM-1 Bispecific Antibody Is a Novel Approach for Treating Multiple Myeloma and Lymphoma.

The cluster of differentiation 38 (CD38) is a well-validated target for treating multiple myeloma. Although anti-CD38 mAbs have demonstrated outstanding initial responses in patients with multiple myeloma, nearly all patients eventually develop resistance and relapse. In addition, currently approved CD38 targeting therapies have failed to show monotherapy efficacy in lymphomas, where CD38 expression is present but at lower levels. To effectively target CD38 on tumor cells, we generated an antibody-dependent cellular cytotoxicity (ADCC) enhanced bispecific CD38 x intercellular cell adhesion molecule 1 (ICAM-1) antibody, VP301. This bispecific antibody targets unique epitopes on CD38 and ICAM-1 on tumor cells with reduced red blood cell binding compared with the benchmark CD38 antibody daratumumab. VP301 demonstrated potent ADCC and antibody-dependent cellular phagocytosis activities on a selected set of myeloma and lymphoma cell lines even those with low CD38 expression. In an ex vivo drug sensitivity assay, we observed responses to VP301 in multiple myeloma primary samples from relapsed/refractory patients. Moreover, VP301 demonstrated potent tumor inhibition activities in in vivo myeloma and lymphoma models. Interestingly, combination of VP301 with the immunomodulatory drug, lenalidomide, led to synergistic antitumor growth activity in an in vivo efficacy study. In conclusion, the CD38 x ICAM-1 bispecific antibody VP301 demonstrated promising efficacy and specificity toward CD38+ and ICAM-1+ tumor cells and represents a novel approach for treating multiple myeloma and lymphoma.

CD46-ADC Reduces the Engraftment of Multiple Myeloma Patient-Derived Xenografts.

An antibody-drug conjugate (ADC) targeting CD46 conjugated to monomethyl auristatin has a potent anti-myeloma effect in cell lines in vitro and in vivo, and patient samples treated ex vivo. Here, we tested if CD46-ADC may have the potential to target MM-initiating cells (MM-ICs). CD46 expression was measured on primary MM cells with a stem-like phenotype. A patient-derived xenograft (PDX) model was implemented utilizing implanted fetal bone fragments to provide a humanized microenvironment. Engraftment was monitored via serum human light chain ELISA, and at sacrifice via bone marrow and bone fragment flow cytometry. We then tested MM regeneration in PDX by treating mice with CD46-ADC or the nonbinding control-ADC. MM progenitor cells from patients that exhibit high aldehyde dehydrogenase activity also have a high expression of CD46. In PDX, newly diagnosed MM patient samples engrafted significantly more compared to relapsed/refractory samples. In mice transplanted with newly diagnosed samples, CD46-ADC treatment showed significantly decreased engraftment compared to control-ADC treatment. Our data further support the targeting of CD46 in MM. To our knowledge, this is the first study to show preclinical drug efficacy in a PDX model of MM. This is an important area for future study, as patient samples but not cell lines accurately represent intratumoral heterogeneity.

CD38 antibody re-treatment in daratumumab-refractory multiple myeloma after time on other therapies.

Monoclonal antibodies targeting CD38 are important for treatment of both newly diagnosed and relapsed multiple myeloma (MM). Daratumumab and isatuximab are anti-CD38 antibodies with the US Food and Drugs Administration approval in multiple different combinations. Despite good initial efficacy, patients inevitably develop drug resistance. Whether patients can be effectively re-treated with these antibodies in subsequent lines of therapy is unclear. Thus far, studies have mostly been limited to clinical retrospectives with short washout periods. To answer whether patients regain sensitivity after longer washouts, we used ex vivo sensitivity testing to isolate the anti-CD38 antibody-specific cytotoxicity in samples obtained from patients who had been exposed to and then off daratumumab for up to 53 months. MM cells from patients who had been off daratumumab for >1 year showed greater sensitivity than those with <1 year, although they still were less sensitive than those who were daratumumab naïve. CD38 expression on MM cells gradually recovered, although, again, not to the level of anti-CD38 antibody-naïve patients. Interestingly, low MM CD38 explained only 45% of cases identified to have daratumumab resistance. With clinical follow-up, we found ex vivo sensitivity predicted subsequent clinical response but CD38 overexpression did not. Patients clinically re-treated with anti-CD38 antibodies had <6 months of clinical benefit, but 1 patient who was daratumumab exposed but not refractory achieved complete response lasting 13 months. We conclude that transient efficacy can be achieved by waiting 1 year before CD38 antibody rechallenge, but this approach may be best used as a bridge to, or after, chimeric antigen receptor T-cell therapy.

Lenalidomide-induced autoimmune enteropathy complicating treatment of multiple myeloma with concurrent systemic mastocytosis.

Lenalidomide is an IMiD drug which has been associated with a variety of potential immune related complications. We describe the case of a patient with newly diagnosed multiple myeloma along with a history of systemic mastocytosis who developed evidence of an autoimmune enteropathy shortly after initiating lenalidomide based therapy.

Harnessing the T Cell to Treat Multiple Myeloma: Dawn of a New Therapeutic Paradigm.

Multiple myeloma is an incurable hematologic malignancy. The typical disease course for myeloma patients is characterized by initial response to treatment followed by eventual development of resistance. Subsequent cycles of remission and relapse proceed as long as patients have new lines of therapy available to them. This reality has prompted development of many novel immunotherapeutics. Many of these drugs exploit the cytotoxic capabilities of the patients' own T cells, effectively redirecting them to myeloma cells that are otherwise evading immune attack. Approaches including CAR T cell therapy and bispecific antibodies have displayed impressive efficacy in clinical trials for myeloma patients. This review examines the different approaches that utilize T cells in multiple myeloma therapy and investigates the benefits and risks of these exciting new strategies.

Emerging Therapeutic Strategies to Overcome Drug Resistance in Multiple Myeloma.

Multiple myeloma is a malignant plasma cell neoplasm that remains incurable and is ultimately fatal when patients acquire multi-drug resistance. Thus, advancing our understanding of the mechanisms behind drug resistance in multi-relapsed patients is critical for developing better strategies to extend their lifespan. Here, we review the understanding of resistance to the three key drug classes approved for multiple myeloma treatment: immunomodulatory drugs, proteasome inhibitors, and monoclonal antibodies. We consider how the complex, heterogenous biology of multiple myeloma may influence the acquisition of drug resistance and reflect on the gaps in knowledge where additional research is needed to improve our treatment approaches. Fortunately, many agents are currently being evaluated preclinically and in clinical trials that have the potential to overcome or delay drug resistance, including next-generation immunomodulatory drugs and proteasome inhibitors, novel small molecule drugs, chimeric antigen receptor T cells, antibody-drug conjugates, and bispecific antibodies. For each class, we discuss the potential of these strategies to overcome resistance through modifying agents within each class or new classes without cross-resistance to currently available drugs.

Exploiting Protein Translation Dependence in Multiple Myeloma with Omacetaxine-Based Therapy.

PURPOSE: The prognosis of patients with multiple myeloma who are resistant to proteasome inhibitors, immunomodulatory drugs (IMiD), and daratumumab is extremely poor. Even B-cell maturation antigen-specific chimeric antigen receptor T-cell therapies provide only a temporary benefit before patients succumb to their disease. In this article, we interrogate the unique sensitivity of multiple myeloma cells to the alternative strategy of blocking protein translation with omacetaxine. EXPERIMENTAL DESIGN: We determined protein translation levels (n = 17) and sensitivity to omacetaxine (n = 51) of primary multiple myeloma patient samples. Synergy was evaluated between omacetaxine and IMiDs in vitro, ex vivo, and in vivo. Underlying mechanism was investigated via proteomic analysis. RESULTS: Almost universally, primary patient multiple myeloma cells exhibit >2.5-fold increased rates of protein translation compared with normal marrow cells. Ex vivo treatment with omacetaxine resulted in >50% reduction in viable multiple myeloma cells. In this cohort, high levels of translation serve as a biomarker for patient multiple myeloma cell sensitivity to omacetaxine. Unexpectedly, omacetaxine demonstrated synergy with IMiDs in multiple myeloma cell lines in vitro. In addition, in an IMiD-resistant relapsed patient sample, omacetaxine/IMiD combination treatment resensitized the multiple myeloma cells to the IMiD. Proteomic analysis found that the omacetaxine/IMiD combination treatment produced a double-hit on the IRF4/c-MYC pathway, which is critical to multiple myeloma survival. CONCLUSIONS: Overall, protein translation inhibitors represent a potential new drug class for myeloma treatment and provide a rationale for conducting clinical trials with omacetaxine alone and in combination with IMiDs for patients with relapsed/refractory multiple myeloma.

Potent Activity of an Anti-ICAM1 Antibody-Drug Conjugate against Multiple Myeloma.

PURPOSE: New therapies have changed the outlook for patients with multiple myeloma, but novel agents are needed for patients who are refractory or relapsed on currently approved drug classes. Novel targets other than CD38 and BCMA are needed for new immunotherapy development, as resistance to daratumumab and emerging anti-BCMA approaches appears inevitable. One potential target of interest in myeloma is ICAM1. Naked anti-ICAM1 antibodies were active in preclinical models of myeloma and safe in patients, but showed limited clinical efficacy. Here, we sought to achieve improved targeting of multiple myeloma with an anti-ICAM1 antibody-drug conjugate (ADC). EXPERIMENTAL DESIGN: Our anti-ICAM1 human mAb was conjugated to an auristatin derivative, and tested against multiple myeloma cell lines in vitro, orthotopic xenografts in vivo, and patient samples ex vivo. The expression of ICAM1 was also measured by quantitative flow cytometry in patients spanning from diagnosis to the daratumumab-refractory state. RESULTS: The anti-ICAM1 ADC displayed potent anti-myeloma cytotoxicity in vitro and in vivo. In addition, we have verified that ICAM1 is highly expressed on myeloma cells and shown that its expression is further accentuated by the presence of bone marrow microenvironmental factors. In primary samples, ICAM1 is differentially overexpressed on multiple myeloma cells compared with normal cells, including daratumumab-refractory patients with decreased CD38. In addition, ICAM1-ADC showed selective cytotoxicity in multiple myeloma primary samples. CONCLUSIONS: We propose that anti-ICAM1 ADC should be further studied for toxicity, and if safe, tested for clinical efficacy in patients with relapsed or refractory multiple myeloma.

Measurement of ex vivo resistance to proteasome inhibitors, IMiDs, and daratumumab during multiple myeloma progression.

The oncogenic drivers and progression factors in multiple myeloma (MM) are heterogeneous and difficult to target therapeutically. Many different MM drugs have emerged, however, that attack various phenotypic aspects of malignant plasma cells. These drugs are administered in numerous, seemingly interchangeable combinations. Although the availability of many treatment options is useful, no clinical test capable of optimizing and sequencing the treatment regimens for an individual patient is currently available. To overcome this problem, we developed a functional ex vivo approach to measure patients' inherent and acquired drug resistance. This method, which we termed myeloma drug sensitivity testing (My-DST), uses unselected bone marrow mononuclear cells with a panel of drugs in clinical use, followed by flow cytometry to measure myeloma-specific cytotoxicity. We found that using whole bone marrow cultures helped preserve primary MM cell viability. My-DST was used to profile 55 primary samples at diagnosis or at relapse. Sensitivity or resistance to each drug was determined from the change in MM viability relative to untreated control samples. My-DST identified progressive loss of sensitivity to immunomodulatory drugs, proteasome inhibitors, and daratumumab through the disease course, mirroring the clinical development of resistance. Prospectively, patients' ex vivo drug sensitivity to the drugs subsequently received was sensitive and specific for clinical response. In addition, treatment with <2 drugs identified as sensitive by My-DST led to inferior depth and duration of clinical response. In summary, ex vivo drug sensitivity is prognostically impactful and, with further validation, may facilitate more personalized and effective therapeutic regimens.

Disruption of IRE1α through its kinase domain attenuates multiple myeloma.

Multiple myeloma (MM) arises from malignant immunoglobulin (Ig)-secreting plasma cells and remains an incurable, often lethal disease despite therapeutic advances. The unfolded-protein response sensor IRE1α supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may co-opt the IRE1α-XBP1s pathway; however, the validity of IRE1α as a potential MM therapeutic target is controversial. Genetic disruption of IRE1α or XBP1s, or pharmacologic IRE1α kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice and augmented efficacy of two established frontline antimyeloma agents, bortezomib and lenalidomide. Mechanistically, IRE1α perturbation inhibited expression of key components of the endoplasmic reticulum-associated degradation machinery, as well as secretion of Ig light chains and of cytokines and chemokines known to promote MM growth. Selective IRE1α kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138- cells derived from bone marrows of newly diagnosed or posttreatment-relapsed MM patients, in both US- and European Union-based cohorts. Effective IRE1α inhibition preserved glucose-induced insulin secretion by pancreatic microislets and viability of primary hepatocytes in vitro, as well as normal tissue homeostasis in mice. These results establish a strong rationale for developing kinase-directed inhibitors of IRE1α for MM therapy.

Phase II study of carfilzomib and dexamethasone therapy for newly diagnosed multiple myeloma.

Carfilzomib and dexamethasone (Kd) has significant activity in relapsed and refractory multiple myeloma. Kd has not previously been evaluated in newly diagnosed multiple myeloma (NDMM). We report a single-arm phase 2 study of 72 patients with NDMM to evaluate the efficacy and tolerability of Kd induction. Carfilzomib was administered in two dosing cohorts with dosing of 20/45 mg/m2 in the first 25 patients and 20/56 mg/m2 in the subsequent 47 patients. Carfilzomib was administered on days 1, 2, 8, 9, 15 and 16 of a 28-day cycle, dexamethasone 20 mg was administered orally on days 1, 2, 8, 9, 15, 16, 22 and 23. Treatment was continued to maximum response, progression of disease, or regimen intolerability. Endpoints included overall response rate (ORR), regimen toxicity and impact of carfilzomib on CD34+ stem cell collection yield. Sixty-five pts achieved at least a partial response (PR) for an ORR of 90%. The maximum response achieved was complete response or better in 5 (7%), very good partial response (VGPR) in 42 (58%), PR in 18 (25%) and stable disease in 7 pts (10%). Toxicities were predominantly low grade with 547 grade 1/2 adverse events and 44 grade ≥3 events. The rate of grade ≥3 cardiovascular adverse events was 11.1% with eight observed events. The activity of Kd described represents the highest rate of overall response and ≥VGPR for any 2-agent combination in NDMM reported to date. Kd demonstrated a safety profile consistent with previously reported carfilzomib studies.

Targeting CD46 for both adenocarcinoma and neuroendocrine prostate cancer.

Although initially responsive to androgen signaling inhibitors (ASIs), metastatic castration-resistant prostate cancer (mCRPC) inevitably develops and is incurable. In addition to adenocarcinoma (adeno), neuroendocrine prostate cancer (NEPC) emerges to confer ASI resistance. We have previously combined laser capture microdissection and phage antibody display library selection on human cancer specimens and identified novel internalizing antibodies binding to tumor cells residing in their tissue microenvironment. We identified the target antigen for one of these antibodies as CD46, a multifunctional protein that is best known for negatively regulating the innate immune system. CD46 is overexpressed in primary tumor tissue and CRPC (localized and metastatic; adeno and NEPC), but expressed at low levels on normal tissues except for placental trophoblasts and prostate epithelium. Abiraterone- and enzalutamide-treated mCRPC cells upregulate cell surface CD46 expression. Genomic analysis showed that the CD46 gene is gained in 45% abiraterone-resistant mCRPC patients. We conjugated a tubulin inhibitor to our macropinocytosing anti-CD46 antibody and showed that the resulting antibody-drug conjugate (ADC) potently and selectively kills both adeno and NEPC cell lines in vitro (sub-nM EC50) but not normal cells. CD46 ADC regressed and eliminated an mCRPC cell line xenograft in vivo in both subcutaneous and intrafemoral models. Exploratory toxicology studies of the CD46 ADC in non-human primates demonstrated an acceptable safety profile. Thus, CD46 is an excellent target for antibody-based therapy development, which has potential to be applicable to both adenocarcinoma and neuroendocrine types of mCRPC that are resistant to current treatment.

Cell-type specific potent Wnt signaling blockade by bispecific antibody.

Cell signaling pathways are often shared between normal and diseased cells. How to achieve cell type-specific, potent inhibition of signaling pathways is a major challenge with implications for therapeutic development. Using the Wnt/ß-catenin signaling pathway as a model system, we report here a novel and generally applicable method to achieve cell type-selective signaling blockade. We constructed a bispecific antibody targeting the Wnt co-receptor LRP6 (the effector antigen) and a cell type-associated antigen (the guide antigen) that provides the targeting specificity. We found that the bispecific antibody inhibits Wnt-induced reporter activities with over one hundred-fold enhancement in potency, and in a cell type-selective manner. Potency enhancement is dependent on the expression level of the guide antigen on the target cell surface and the apparent affinity of the anti-guide antibody. Both internalizing and non-internalizing guide antigens can be used, with internalizing bispecific antibody being able to block signaling by all ligands binding to the target receptor due to its removal from the cell surface. It is thus feasible to develop bispecific-based therapeutic strategies that potently and selectively inhibit signaling pathways in a cell type-selective manner, creating opportunity for therapeutic targeting.

Monoclonal Antibodies in Multiple Myeloma: A New Wave of the Future.

In 2015, 2 monoclonal antibodies were approved for the treatment of relapsed or refractory multiple myeloma (RRMM), elotuzumab and daratumumab. Elotuzumab is a monoclonal IgG-κ antibody directed against SLAMF7 (signaling lymphocytic activation molecule F7), a cell surface receptor involved in natural killer cell activation. Daratumumab is a monoclonal IgG-κ antibody that binds to CD38, a transmembrane protein found on the surface of myeloma cells and responsible for cellular adhesion and ectoenzymatic activity. Both elotuzumab and daratumumab act through recruitment of the immune system to enhance cellular cytotoxicity directed against myeloma cells. Elotuzumab requires lenalidomide and dexamethasone combined to enhance progression-free survival in patients with RRMM, and daratumumab has both single-agent and combination activity with either lenalidomide or the proteasome inhibitor bortezomib in RRMM. The adverse effect profile of both agents mainly consists of allergic-type infusion reactions. Other considerations for monoclonal antibody use in the treatment of MM include the potential for interference in serum protein electrophoresis testing and cross-reactivity of daratumumab with CD38 present on red blood cells. In the present report, we discussed the clinical development of daratumumab and elotuzumab and newer immunologic approaches to the treatment of MM.

Antibody-drug conjugate targeting CD46 eliminates multiple myeloma cells.

Multiple myeloma is incurable by standard approaches because of inevitable relapse and development of treatment resistance in all patients. In our prior work, we identified a panel of macropinocytosing human monoclonal antibodies against CD46, a negative regulator of the innate immune system, and constructed antibody-drug conjugates (ADCs). In this report, we show that an anti-CD46 ADC (CD46-ADC) potently inhibited proliferation in myeloma cell lines with little effect on normal cells. CD46-ADC also potently eliminated myeloma growth in orthometastatic xenograft models. In primary myeloma cells derived from bone marrow aspirates, CD46-ADC induced apoptosis and cell death, but did not affect the viability of nontumor mononuclear cells. It is of clinical interest that the CD46 gene resides on chromosome 1q, which undergoes genomic amplification in the majority of relapsed myeloma patients. We found that the cell surface expression level of CD46 was markedly higher in patient myeloma cells with 1q gain than in those with normal 1q copy number. Thus, genomic amplification of CD46 may serve as a surrogate for target amplification that could allow patient stratification for tailored CD46-targeted therapy. Overall, these findings indicate that CD46 is a promising target for antibody-based treatment of multiple myeloma, especially in patients with gain of chromosome 1q.

The development of potential antibody-based therapies for myeloma.

With optimal target antigen selection antibody-based therapeutics can be very effective agents for hematologic malignancies, but none have yet been approved for myeloma. Rituximab and brentuximab vedotin are examples of success for the naked antibody and antibody-drug conjugate classes, respectively. Plasma cell myeloma is an attractive disease for antibody-based targeting due to target cell accessibility and the complementary mechanism of action with approved therapies. Initial antibodies tested in myeloma were disappointing. However, recent results from targeting well-characterized antigens have been more encouraging. In particular, the CD38 and CD138 targeted therapies are showing single-agent activity in early phase clinical trials. Here we will review the development pipeline for naked antibodies and antibody-drug conjugates for myeloma. There is clear clinical need for new treatments, as myeloma inevitably becomes refractory to standard agents. The full impact is yet to be established, but we are optimistic that the first FDA-approved antibody therapeutic(s) for this disease will emerge in the near future.

BCR-ABL SH3-SH2 domain mutations in chronic myeloid leukemia patients on imatinib.

Point mutations in the kinase domain of BCR-ABL are the most common mechanism of drug resistance in chronic myeloid leukemia (CML) patients treated with ABL kinase inhibitors, including imatinib. It has also been shown in vitro that mutations outside the kinase domain in the neighboring linker, SH2, SH3, and Cap domains can confer imatinib resistance. In the context of ABL, these domains have an autoinhibitory effect on kinase activity, and mutations in this region can activate the enzyme. To determine the frequency and relevance to resistance of regulatory domain mutations in CML patients on imatinib, we screened for such mutations in a cohort of consecutive CML patients with various levels of response. Regulatory domain mutations were detected in 7 of 98 patients, whereas kinase domain mutations were detected in 29. One mutation (T212R) conferred in vitro tyrosine kinase inhibitor resistance and was associated with relapse, whereas most other mutations did not affect drug sensitivity. Mechanistic studies showed that T212R increased the activity of ABL and BCR-ABL and that T212R-induced resistance may be partially the result of stabilization of an active kinase conformation. Regulatory domain mutations are uncommon but may explain resistance in some patients without mutations in the kinase domain.