Mutations In Bruton's Tyrosine Kinase Research Articles

Abstract 4391: Overcoming primary ibrutinib resistance in mantle cell lymphoma

Abstract Mantle cell lymphoma (MCL) is a rare and incurable subtype of B-cell lymphoma. In a phase II study of ibrutinib in MCL patients, most of the patients responded and had long durable remissions; however, 22.7% of patients were considered to be non-responsive to ibrutinib, and an additional 22/110 patients displayed initial positive responses to ibrutinib but also experienced disease progression within 12 months of treatment, with both responses classified as primary ibrutinib resistant. Therefore, understanding the mechanisms mediating primary ibrutinib resistance may identify new prognostic and predictive markers and potential therapeutic targets. Bruton's tyrosine kinase (BTK) plays an important role in B-cell development, activation, and differentiation. Upon B-cell receptor (BCR) activation, BTK is phosphorylated and activated by SYK. Phosphoinositide 3-kinase (PI3K) is recruited to the BCR, and the induction of these signaling activities leads to the downstream activation of multiple effector proteins, including nuclear factor-kB (NF-kB), AKT, RAS, mTOR and mitogen-activated protein kinase (MAPK). BTK and PI3K have been shown to function independently to mediate BCR signaling, suggesting that PI3K signaling activities may underlie ibrutinib resistance independently of BTK. PI3K has also been associated with primary ibrutinib resistance. To further elucidate the mechanisms underlying ibrutinib resistance, we conferred primary ibrutinib resistance using both MCL cell lines and MCL-bearing patient-derived xenograft (PDX) mouse models and used whole exome sequencing (WES) and reverse phase protein analysis (RPPA) to identify any genetic and expression changes associated with primary ibrutinib resistance. WES did not reveal any mutations in BTK or within the proximal BCR pathway, consistent with WES data on primary ibrutinib resistant MCL cases. RPPA analysis showed a significant increase in the PI3K/AKT/mTOR/MCL-1 compensatory pathway component levels in ibrutinib-resistant cell lines when compared with their parental cells as well as ibrutinib-resistant PDXs. To determine whether inhibiting these pathways would overcome primary ibrutinib resistance, we tested various therapeutic combinations targeting these pathways. Ibrutinib plus the PI3K inhibitor idelalisib, the AKT inhibitor ACP-319, the mTOR inhibitors AZD8055 or BEZ235 as well as the proteasome inhibitor carfilzomib inhibited tumor growth in vitro and in vivo in the PDX mouse models, demonstrating that targeting these alternative pathways may overcome ibrutinib resistance. This work strongly suggests that targeting the PI3K/AKT/mTOR/MCL-1 compensatory pathway successfully inhibits the viability of ibrutinib-resistant MCL tumor cells both in vitro and in vivo and identified potential therapies that can be used to treat ibrutinib-resistant patients in clinical practice. Citation Format: Leo Zhang, Lan Pham, Hui Zhang, Jingmeng Xie, Wenjing Tao, Taylor Bell, Zhihong Chen, Krystle Nomie, Bingliang Fang, Michael Wang. Overcoming primary ibrutinib resistance in mantle cell lymphoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4391.

Myeloid-Derived Suppressor Cells Express Bruton's Tyrosine Kinase and Can Be Depleted in Tumor-Bearing Hosts by Ibrutinib Treatment.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of immature myeloid cells that expand in tumor-bearing hosts in response to soluble factors produced by tumor and stromal cells. MDSC expansion has been linked to loss of immune effector cell function and reduced efficacy of immune-based cancer therapies, highlighting the MDSC population as an attractive therapeutic target. Ibrutinib, an irreversible inhibitor of Bruton's tyrosine kinase (BTK) and IL2-inducible T-cell kinase (ITK), is in clinical use for the treatment of B-cell malignancies. Here, we report that BTK is expressed by murine and human MDSCs, and that ibrutinib is able to inhibit BTK phosphorylation in these cells. Treatment of MDSCs with ibrutinib significantly impaired nitric oxide production and cell migration. In addition, ibrutinib inhibited in vitro generation of human MDSCs and reduced mRNA expression of indolamine 2,3-dioxygenase, an immunosuppressive factor. Treatment of mice bearing EMT6 mammary tumors with ibrutinib resulted in reduced frequency of MDSCs in both the spleen and tumor. Ibrutinib treatment also resulted in a significant reduction of MDSCs in wild-type mice bearing B16F10 melanoma tumors, but not in X-linked immunodeficiency mice (XID) harboring a BTK mutation, suggesting that BTK inhibition plays an important role in the observed reduction of MDSCs in vivo Finally, ibrutinib significantly enhanced the efficacy of anti-PD-L1 (CD274) therapy in a murine breast cancer model. Together, these results demonstrate that ibrutinib modulates MDSC function and generation, revealing a potential strategy for enhancing immune-based therapies in solid malignancies. Cancer Res; 76(8); 2125-36. ©2016 AACR.

X-Linked Agammagobulinemia in a Large Series of North African Patients: Frequency, Clinical Features and Novel BTK Mutations.

X-linked agammagobulinemia (XLA) is a primary immunodeficiency caused by Bruton's tyrosine kinase (BTK) gene defect. XLA patients have absent or reduced number of peripheral B cells and a profound deficiency in all immunoglobulin isotypes. This multicenter study reports the clinical, immunological and molecular features of Bruton's disease in 40 North African male patients. Fifty male out of 63 (male and female) patients diagnosed with serum agammaglobulinemia and non detectable to less than 2% peripheral B cells were enrolled. The search for BTK gene mutations was performed for all of them by genomic DNA amplification and Sanger sequencing. We identified 33 different mutations in the BTK gene in 40 patients including 12 missense mutations, 6 nonsense mutations, 6 splice-site mutations, 5 frameshift, 2 large deletions, one complex mutation and one in-frame deletion. Seventeen of these mutations are novel. This large series shows a lower frequency of XLA among male patients from North Africa with agammaglobulinemia and absent to low B cells compared with other international studies (63.5% vs. 85%). No strong evidence for genotype-phenotype correlation was observed. This study adds to other reports from highly consanguineous North African populations, showing lower frequency of X-linked forms as compared to AR forms of the same primary immunodeficiency. Furthermore, a large number of novel BTK mutations were identified and could further help identify carriers for genetic counseling.

High Sensitivity Testing Shows Multiclonal Mutations in Patients with CLL Treated with BTK Inhibitor and Lack of Mutations in Ibrutinib-Naive Patients

Background: Patients with chronic lymphocytic leukemia (CLL) that develop resistance to Bruton's tyrosine kinase (BTK) inhibitors are typically positive for mutations in BTK or phospholipase c gamma 2 (PLCγ2). Mutations in BTK at the C481S hotspot alter the active site of the mutant BTK to the effect that Ibrutinib is reversibly bound. PLCγ2 is downstream of BTK in the B-cell signaling pathway; mutations in PLCγ2 at either of the R665W, L845F, or S707Y hotspots result in a constitutively activated PLCγ2. In order to better understand the development of these resistance mechanisms in patients with CLL, we developed a high sensitivity (HS) assay utilizing branched and locked nucleic acids (BNA and LNA, respectively). We used this high sensitivity assay in combination with Sanger sequencing and next generation sequencing (NGS) and tested cellular DNA and cell free DNA (cf-DNA) from patients with CLL.Methods: Using custom BNA or LNA oligos in a wild-type blocking polymerase chain reaction, then sequencing by Sanger and NGS methods, we achieved 100x greater sensitivity than Sanger. Sanger sequencing was capable of detecting <1 mutant allele in background of 1000 wild-type alleles (1:1000). Similar sensitivity was achieved with HS NGS. The assay is designed to cover BTK and PLCγ2 hotspots. Using this assay we tested peripheral blood samples from 44 Ibrutinib-naïve patients (Ib-) with CLL and 7 samples from CLL patients being treated with Ibrutinib (Ib+), that showed clinical evidence of disease progression. The same wild-type blocking was also used in NGS approach for confirmation. We performed wild-type blocking in a Nextera Rapid Capture Enrichment workflow for our custom 315 gene panel.Results: No BTK or PLCγ2 mutations were detected in any of the 44 ibrutinib-naïve CLL patients. In contrast, all (N=7) tested patients with progressive disease on Ibrutinib showed one or more mutation in BTK or PLCγ2 using the HS method. Without the HS testing only 4 patients (57%) showed a mutation in BTK or PLCγ2. Two patients showed multiple mutant clones. One patient with double mutations in PLCγ2 (R665W and L845F) also showed triple independent mutations in BTK at codon C481 with HS testing. These mutations give rise to two distinct mutant proteins C481R (TGC>CGC) and C481S (TGC>AGC and TGC>TCC). NGS analysis confirmed that the the three BTK mutations are in three independent clones A second patient showed initially a mutation in BTK (C481S), but subsequent sample showed a mutation in PLCg2 (R665W), in addition to the BTK mutation. All mutations detected in the peripheral blood cells were also detectable in cell-free DNA (cfDNA) using HS testing. However, without using HS testing, a BTK mutation was detected in cfDNA from a patient and this mutation was not detectable when cellular DNA was used.Conclusions: Our data suggests that ibrutinib-naïve patients with CLL do not have BTK or PLCγ2 mutations even when a highly sensitive assay is used. Emerging BTK or PLCγ2 mutant clones can be seen after therapy with the possibility of multiple clones emerging at the same time and may involve both BTK and PLCγ2 genes in the same patient. Furthermore, testing cfDNA is not only as informative as cellular DNA, but might show mutations earlier than cellular DNA. This may have clinical relevance in patients with lymphoma when only few lymphoma cells are in circulation. [Display omitted] DisclosuresWiestner:Pharmacyclics: Research Funding.

BET Protein Bromodomain Antagonist-Based Combinations Exert Synergistic Pre-Clinical Activity Against Ibrutinib-Sensitive or -Resistant Human Mantle Cell Lymphoma (MCL) Cells

Mantle Cell Lymphoma (MCL) exhibits genetic alterations in p53, CDK4, CDKN2A, MYC, BCL2, B cell receptor (BCR) and NFkB signaling, which confer a cell autonomous, pro-growth and pro-survival advantage on MCL cells. Treatment with the Bruton's Tyrosine Kinase (BTK) inhibitor ibrutinib inhibits BCR and NFκB activity, mediating growth inhibition and apoptosis of MCL cells. Although ibrutinib induces impressive clinical responses in patients with MCL, long-term durable remissions and cure remain elusive. Ibrutinib-resistant MCL cells exhibit mutations in TRAF2/3 and MAP3K14 (NIK), activating the alternative NFκB signaling. Additionally, a cysteine-to-serine C481S mutation in BTK at the binding site of ibrutinib, which confers resistance to ibrutinib, has also been documented in MCL that relapsed while on ibrutinib. These observations suggest that ibrutinib-resistant MCL cells remain dependent for growth and survival on the NFκB-activated gene-expressions. In the ibrutinib-refractory primary MCL cells, mutations in MLL2, CREBBP, PIM1 and ERB4 were also detected. Our findings have shown that the BET (bromodomain and extra-terminal) protein BRD4 antagonists (BA), e.g., JQ1, transcriptionally repress the levels of MCL-relevant oncogenes BCL-2, c-Myc, cyclin D1, CDK6 and PIM1, while inducing the levels of HEXIM1, p21 and p27 in the MCL cells. Consistent with the reported findings that BRD4 is essential for the transcriptional activity of NFkB, JQ1 treatment also attenuated the nuclear RelA and inhibited the expression of several NFkB target genes, including XIAP, IkBα, cFLIP, cIAP2, as well as BTK. Collectively, this resulted in growth arrest and apoptosis of the cultured (MO2058, Mino, Z138 and JeKo-1) and primary MCL (pMCL) cells. We also demonstrate that compared to ibrutinib alone, co-treatment with JQ1 and ibrutinib markedly inhibits pBTK, BTK, pPLCγ2, pAKT, PIM1 and nuclear RelA levels, as well as synergistically induced apoptosis of the cultured and pMCL cells with combination indices (CI) of less than 1.0 by the median dose effect isobologram analysis. Following tail-vein infusion and engraftment of Mino cells in the bone marrow and spleen, co-treatment with JQ1 and ibrutinib, versus each agent alone, significantly improved the survival of NOD/SCID mice. By exposing parental MCL Mino or MO2058 cells to escalating levels of ibrutinib, we isolated Ibrutinib-resistant Mino/IR (>13-fold resistant) and MO2058/IR cells (>8-fold resistant), respectively. Sequencing of the BTK gene did not reveal any mutation in the BTK gene (e.g. C481S) in the ibrutinib-resistant cells. Notably, compared to Mino, Mino/IR cells expressed higher protein levels of BTK, AKT, CDK6, PIM1, XIAP, Bcl-xL and BCL2 levels, while simultaneously displaying lower NOXA, PUMA and p21 levels. Importantly, treatment with JQ1 inhibited the suspension culture growth of Mino/IR cells. JQ1 treatment also induced similar level of apoptosis in Mino/IR, as compared to Mino cells. JQ1-induced apoptosis of Mino/IR cells was associated with a reduction in the nuclear levels of RelA, as well as inhibition of the mRNA levels of BTK, CDK6, and BCL2. In contrast, JQ1 treatment induced the mRNA expression of BCL2L11 and HEXIM1. JQ1 treatment also markedly attenuated the protein levels of BTK, c-Myc, PIM and CDK6 in Mino/IR cells; however, a more modest inhibition of Bcl-xL, BCL2, and XIAP protein levels, and a lack of effect on the protein levels of AKT was observed. Furthermore, pre-treatment of Mino/IR cells with JQ1 enhanced their sensitivity to ibrutinib. Next, we determined that co-treatment with JQ1 and the BCL2 antagonist ABT-199, palbociclib (a CDK4 and CDK6 inhibitor) or AZD1208 (a PIM1 & 2 kinase inhibitor) was synergistically lethal against Mino and Mino/IR cells (CI < 1.0). Co-treatment with JQ1 and ABT199, AZD1208 or palbociclib was also synergistically lethal against pMCL cells, but not against the normal B or hematopoietic cells. These findings demonstrate that treatment with the BA JQ1 sensitizes MCL cells to not only ibrutinib but also to BCL2, PIM1 & 2 and CDK4/6 antagonists. Furthermore, co-treatment with BA and BCL2, PIM1 & 2 or CDK4/6 antagonist are promising combination therapies for testing against ibrutinib-sensitive or ibrutinib-resistant MCL cells. DisclosuresWang:Pharmacyclics, Janssen, Celgene, Oncopep, Kite, Juno: Research Funding; Janssen: Honoraria.

Synergistic Co-Targeting of BTK and BCL2 in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL), a B-cell non-Hodgkin lymphoma, remains incurable with current treatment modalities. Bruton tyrosine kinase (BTK), a key component of the early BCR signaling pathway, has emerged as a promising therapeutic target. Ibrutinib, a specific inhibitor that binds covalently to the active site of BTK at cysteine 481, has been approved for the treatment of MCL. In a recent Phase II study, treatment for relapsed or refractory MCL with Ibrutinib alone achieved a response rate of 68%. Although this response rate is striking, approximately one third of patients show primary resistance, and acquired resistance with a C481S mutation in BTK can also develop. To overcome primary and acquired resistance to BTK inhibition, a combinatory strategy that targets multiple pathways is needed.Here, we focused on the BCL2 anti-apoptotic pathway. Our previous study established FBXO10 as the E3 ubiquitin ligase that targets BCL2 for proteasomal degradation and revealed reduced levels of FBXO10 expression in MCL cell lines. To examine BCL2 and FBXO10 expression in patient samples, we performed immunohistochemical (IHC) analysis in a tissue microarray (TMA) that contained 62 MCL cases. Our data showed high levels of BCL2 expression but no or low FBXO10 expression in MCL cases. Based on this initial expression analysis, we hypothesized that a defect in FBXO10-mediated proteasomal degradation contributes to high BCL2 expression in MCL. To test this hypothesis, we genetically manipulated FBXO10 expression in MCL cell lines. Flow cytometric analysis revealed that overexpression of FBXO10 resulted in reduced BCL2 expression. Inversely, we silenced endogenous FBXO10 by shRNA. Indeed, after blocking protein synthesis by cycloheximide, the amount of BCL2 protein was sustained in these FBXO10 shRNA-expressing cells. These results indicate that BCL2 is the predominantly expressed anti-apoptotic protein in MCL, due to a specific defect in its proteasomal degradation. Inhibition of BCL2 by the specific inhibitor ABT-199 killed cancer cells in culture and also inhibited xenografted tumor growth.BCL2 is a target gene of the BTK/canonical NF-κB signaling pathway. We next analyzed BTK expression in the above TMA and found that BTK was highly expressed and positively correlated with BCL2 expression in these cases. BTK inhibition reduced BCL2 expression as shown in the BCR-dependent cell lines Jeko and Mino. RNA-seq analysis confirmed that a set of anti-apoptotic genes (e.g. BCL2, BCL-XL and DAD1) was downregulated by BTK shRNA. The downregulated genes also included those that are critical for B cell growth and proliferation, such as BCL6, MYC, PIK3CA and BAFF-R. Elevated BCL2, however, can also result from other mechanisms, such as amplification. Indeed, this genomic alteration is present in some MCL cell lines, including Granta-519 and Z138 used for this study. Since these cell lines are insensitive or less sensitive to Ibrutinib, we hypothesized that BCL2 upregulation is a mechanism underlying primary resistance to Ibrutinib, and thus targeting BCL2 by ABT-199 increases or restores sensitivity of these cells to Ibrutinib. The results supported this hypothesis and demonstrated a synergistic effect of ABT-199 and Ibrutinib on growth inhibition of Granta-519 and Z138 cells both in vitro and in vivo.In summary, this study elucidated mechanisms of BCL2 overexpression and association with the BCR/BTK signaling pathway in MCL. Our data provided a mechanistic rationale for co-targeting of these two oncogenic pathways by ABT-199 and Ibrutinib as a new therapeutic strategy in a pre-clinical setting. Most importantly, the findings indicate that this combination has a synergistic effect on the killing of both BCR-dependent and independent MCL cells. Since these two signaling pathways are deregulated in DLBCL and follicular lymphoma, co-targeting by Ibrutinib and ABT-199 may provide an attractive therapeutic strategy for these additional lymphoma patients as well. DisclosuresKahl:Roche/Genentech: Consultancy; Seattle Genetics: Consultancy; Millennium: Consultancy; Cell Therapeutics: Consultancy; Celgene: Consultancy; Infinity: Consultancy; Pharmacyclics: Consultancy; Juno: Consultancy.

Developing Novel Therapeutic Strategies to Overcome Ibrutinib Resistance in Mantle Cell Lymphoma

MCL accounts for 7% of all non-Hodgkin lymphoma cases and is a rare and incurable subtype of B-cell lymphoma. Unfortunately, most MCL patients experience disease progression after frontline therapy, with a median overall survival of approximately 1-2 years after relapse; therefore, novel therapies for MCL are urgently needed. Ibrutinib, a first-in-class, once-daily, oral covalent inhibitor of Bruton's tyrosine kinase (BTK) was recently approved by the FDA to treat this disease. In our prior multiple-center phase II clinical trial, the overall response rate in relapsed/refractory MCL patients was 68%, with a median progression free survival (PFS) of 13.9 months, surpassing the effectiveness of other therapies. Based on our phase II clinical trial, approximately 48% of patients showed no response to ibrutinib or displayed initial positive responses to ibrutinib but also experienced disease progression within 12 months of treatment. Furthermore, the patients who initially show lengthy, durable responses to ibrutinb often acquire resistance and relapse at a median of 17 months. Once patients relapse after ibrutinib treatment, the 1-year survival rate is only 22%; therefore, the vast majority of MCL patients who experience disease progression after ibrutinib treatment will die within 12 months. Overall, the dismal outcomes of these patients demand that novel and pioneering approaches be taken to overcome ibrutinib resistance and improve the cure rate of MCL. To understand the mechanism(s) of acquired resistance for ibrutinib in MCL cells, we have recently generated two ibrutinib-resistant Jeko-1 and PF-1 MCL cell lines. These resistant cells were generated in culture by growing these cells in increasing concentrations of ibrutinib over a period of six months. The ibrutinib-resistant MCL cell lines were at least 8-10-fold more resistant to ibrutinib in comparison to the parental cell lines. The ibrutinib-resistant MCL cell lines were also cross-resistant to another BTK inhibitor, BGB, suggesting that BTK is the specific target in the resistant cell lines. Whole exome sequencing (WES) did not reveal any mutations in BTK or within the proximal BCR pathway, consistent with WES data on primary ibrutinib resistant MCL cases. Next, we conducted proteomics analyses using reverse-phase protein array (RPPA) to examine the protein expression profile of parental and ibrutinib-resistant MCL cell lines. We discovered a significant increase the levels of PI3K/AKT/mTOR/MCL-1 compensatory pathway components in both Jeko-1 and PF-1 ibrutinib-resistant cell lines when compared with their parental cells. Next, we screened for potential therapeutic agents that can overcome ibrutinib resistance. Of the many key agents tested, we discovered that the proteasome inhibitor carfilzomib (CFZ), the Bcl-2 inhibitor ABT-199, and the mTOR inhibitors BEZ and AZD demonstrated the greatest potential to overcome ibrutinib resistance in MCL cells. Interestingly, the ibrutinib-resistant MCL cells developed sensitization to CFZ. We found that the IC50 values of CFZ in the PF-1 ibrutinib-resistant cells were significantly lower (10-fold) in comparison to the IC50 values in the PF-1 parental line, which is consistent with primary MCL cells, where MCL cells derived from ibrutinib-resistant patients are more sensitive to CFZ than ibrutinib-naïve primary MCL cells. These findings are reflected by the function of the 20S proteasome because ibrutinib-resistant MCL cells have higher 20S proteasome activity. In contrast, MCL cell lines and primary MCL cells are highly sensitive to ABT-199 (IC50 < 50 nM) regardless of ibrutinib sensitivity, and ABT-199 is strictly dependent on Bcl-2 protein expression. Furthermore, based on our RPPA data, BEZ and AZD were used to target mTOR to determine whether mTOR inhibition reverses ibrutinib resistance. Our data showed that ibrutinib-resistant MCL cells are more highly resistant to both BEZ and AZD compared with the parental lines. Taken together, our data indicate that CFZ, ABT-199, and mTOR inhibitors have the potential to overcome ibrutinib resistance, and further investigation with in vivo models is warranted to validate our findings. DisclosuresPham:Amgen, Onyx, Millennium: Research Funding. Wang:Celgene: Research Funding.

Outcome of Ibrutinib Treatment by Baseline Genetic Features in Patients with Relapsed or Refractory CLL/SLL with del17p in the Resonate-17 Study

Outcome of Ibrutinib Treatment by Baseline Genetic Features in Patients with Relapsed or Refractory CLL/SLL with del17p in the Resonate-17 Study

Partial reconstitution of humoral immunity and fewer infections in patients with chronic lymphocytic leukemia treated with ibrutinib

AbstractChronic lymphocytic leukemia (CLL) is characterized by immune dysregulation, often including hypogammaglobulinemia, which contributes to a high rate of infections and morbidity. Ibrutinib, a covalent inhibitor of Bruton tyrosine kinase (BTK), inhibits B-cell receptor signaling and is an effective, US Food and Drug Administration (FDA)-approved treatment of CLL. Inactivating germline mutations in BTK cause a severe B-cell defect and agammaglobulinemia. Therefore, we assessed the impact of ibrutinib on immunoglobulin levels, normal B cells, and infection rate in patients with CLL treated with single-agent ibrutinib on a phase 2 investigator-initiated trial. Consistent with previous reports, immunoglobulin G (IgG) levels remained stable during the first 6 months on treatment, but decreased thereafter. In contrast, there were a transient increase in IgM and a sustained increase in IgA (median increase 45% at 12 months, P < .0001). To distinguish the effects on clonal B cells from normal B cells, we measured serum free light chains (FLCs). In κ-clonal CLL cases, clonal (κ) FLCs were elevated at baseline and normalized by 6 months. Nonclonal (λ) FLCs, which were often depressed at baseline, increased, suggesting the recovery of normal B cells. Consistently, we observed normal B-cell precursors in the bone marrow and an increase in normal B-cell numbers in the peripheral blood. Patients with superior immune reconstitution, as defined by an increase in serum IgA of ≥50% from baseline to 12 months, had a significantly lower rate of infections (P = .03). These data indicate that ibrutinib allows for a clinically meaningful recovery of humoral immune function in patients with CLL. This trial was registered at www.clinicaltrials.gov as #NCT01500733.

Prevalence of BTK mutations in male Algerian patterns with agammaglobulinemia and severe B cell lymphopenia

X linked agammaglobulinemia (XLA) is the first described primary immunodeficiency and the most common form of agammaglobulinemia. It is characterized by susceptibility to recurrent infections, profound decrease of all immunoglobulin isotypes and very low level of B lymphocytes in peripheral blood. The disorder is caused by mutations in the Bruton's Tyrosine Kinase (BTK). Nine male patients suspected to have XLA from nine unrelated families were enrolled in this study. We performed sequencing of the BTK gene in all nine patients, and in the patients' relatives when possible. The XLA diagnosis was confirmed for six patients with six different mutations; we identified a novel mutation (c.1522G>A) and five known mutations. One third of nine unrelated patients do not have mutations in BTK and thus likely suffer from autosomal recessive agammaglobulinemia in the setting of consanguinity. Our results support that the autosomal recessive agammaglobulinemia can be more common in Algeria.

Synergistic activity of BET protein antagonist-based combinations in mantle cell lymphoma cells sensitive or resistant to ibrutinib

AbstractMantle cell lymphoma (MCL) cells exhibit increased B-cell receptor and nuclear factor (NF)-κB activities. The bromodomain and extra-terminal (BET) protein bromodomain 4 is essential for the transcriptional activity of NF-κB. Here, we demonstrate that treatment with the BET protein bromodomain antagonist (BA) JQ1 attenuates MYC and cyclin-dependent kinase (CDK)4/6, inhibits the nuclear RelA levels and the expression of NF-κB target genes, including Bruton tyrosine kinase (BTK) in MCL cells. Although lowering the levels of the antiapoptotic B-cell lymphoma (BCL)2 family proteins, BA treatment induces the proapoptotic protein BIM and exerts dose-dependent lethality against cultured and primary MCL cells. Cotreatment with BA and the BTK inhibitor ibrutinib synergistically induces apoptosis of MCL cells. Compared with each agent alone, cotreatment with BA and ibrutinib markedly improved the median survival of mice engrafted with the MCL cells. BA treatment also induced apoptosis of the in vitro isolated, ibrutinib-resistant MCL cells, which overexpress CDK6, BCL2, Bcl-xL, XIAP, and AKT, but lack ibrutinib resistance-conferring BTK mutation. Cotreatment with BA and panobinostat (pan-histone deacetylase inhibitor) or palbociclib (CDK4/6 inhibitor) or ABT-199 (BCL2 antagonist) synergistically induced apoptosis of the ibrutinib-resistant MCL cells. These findings highlight and support further in vivo evaluation of the efficacy of the BA-based combinations with these agents against MCL, including ibrutinib-resistant MCL.

Abstract 136: Evaluation of reversible BTK inhibitors in cell lines with clinically relevant Ibrutinib resistance mutations

Abstract Background Ibrutinib, a small molecule inhibitor that covalently binds Cys481 of Bruton's Tyrosine Kinase (BTK), has recently been approved in Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL). Although the majority of patients respond to this drug, eventually resistance develops. Known causes for resistance include the outgrowth of cancer cells with mutations in either the drug binding pocket of BTK or activating mutations in the downstream effector, PLCγ2. Objective To explore mechanisms of resistance to ibrutinib and evaluate the potential of next generation BTK inhibitors on ibrutinib resistant cancer, we selected for resistance in cultured lymphoma cells. We chose TMD8, a cell line model of diffuse large B-cell lymphoma (DLBCL) that has constitutive B-cell receptor signaling, and REC-1, an ibrutinib sensitive MCL line, to model resistance. Methods TMD8 and REC-1 were cultured in increasing concentrations of ibrutinib to generate resistant cell lines. Genetic changes that may account for resistance were evaluated using whole exome sequencing. Resistant cell lines were evaluated for sensitivity to a small panel of BTK inhibitors. Results We generated a TMD8 derived cell line that is 1000-fold resistant to ibrutinib. Whole exome sequencing revealed a C481Y mutation in BTK. Reversible inhibitors that are not reliant on this site may function as alternative inhibitors of C481 mutant BTK. Ibrutinib-resistant TMD8 cells were tested for response to novel, reversible BTK inhibitors. The potency was similar to or modestly reduced (3 to 10 fold) on the ibrutinib-resistant cells compared to the parental line. In addition, we generated ibrutinib-resistant cells derived from the REC-1 line. Unlike resistance generated in TMD8, REC-1 resistant cells harbor a L527W mutation in BTK and are resistant to all BTK inhibitors that we evaluated. Conclusions C481Y and L527W BTK mutations have been identified in CLL patients that have relapsed after response to ibrutinib, and C481S mutations have been identified in ibrutinib relapsed CLL and MCL patients. Our data suggest that reversible, next generation BTK inhibitors have the potential to be efficacious on ibrutinib relapsed patients that harbor mutations at C481 of BTK. Citation Format: Regina Wai-Yan Choy, Luciana Burton, Wendy Young, James J. Crawford, Elicia Penuel, Lisa D. Belmont. Evaluation of reversible BTK inhibitors in cell lines with clinically relevant Ibrutinib resistance mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 136. doi:10.1158/1538-7445.AM2015-136

Abstract B44: Pharmacologic inhibition with IPI-145 and genetic inhibition of PI3K p110δ antagonizes intrinsic and extrinsic survival signals in chronic lymphocytic leukemia

Abstract Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world, remains an incurable B-cell malignancy. It is characterized by the accumulation of malignant mature B cells in the blood, lymph nodes, spleen and bone marrow. CLL cells display up-regulated B cell receptor (BCR) activation, which maintains B cell survival and proliferation through transmitting microenvironmental stimuli. Due to aberrant regulation of the BCR, CLL cells display constitutively activated survival and proliferation pathways, such as phosphoinositide-3 kinase (PI3K) and Bruton's tyrosine kinase (BTK) pathways. Small molecules that target such kinases in the BCR pathway have shown significant clinical activity in CLL patients. The PI3K p110δ inhibitor, idelalisib, was granted a breakthrough therapy designation by the FDA in relapsed CLL and the BTK inhibitor, ibrutinib, has received approval by FDA for treatment of relapsed CLL. However, patients still relapse on these therapies. Here we use pharmacologic and genetic approaches to further characterize the role of PI3K signaling in the leukemia pathogenesis in the CLL cell and in the microenvironment. We describe that a PI3K p110δ and p110γ inhibitor, IPI-145, which is in late stage clinical development, attenuates pro-survival signals in the OSU-CLL cell line and primary human and murine CLL cells and promotes apoptosis and downstream pathway inactivation in primary human and murine CLL cells in a dose- and time-dependent fashion. To examine the cytotoxicity of IPI-145 in normal immune cells, we incubated whole blood from CLL patients with 0.25-5 μM IPI-145 for 48 hours and analyzed by flow cytometry for absolute count of live CD3+ T cells, CD56+ NK cells and CD19+ B cells. T cells and NK cells were sensitive to IPI-145, displaying about 20% decrease in viability at concentrations greater than 0.5μM, however the B cell population showed about 50% decrease in viability. To specifically examine normal B cells, we isolated CD19+ B cells from healthy volunteer blood and incubated with 1 μM IPI-145 for 48 hours and observed no cytotoxicity, despite observing a significant decrease in CLL cells viability under the same conditions. Additionally, IPI-145 is highly effective at reducing downstream PI3K signaling in a B cell line with the ibrutinib resistance conferring BTK C481S mutation. Genetically we show that the PI3K p110δ-inactivating and the TCL1 leukemia murine models can be utilized to further explore the differential role of PI3K p110δ in leukemic cell and microenvironment. Our study indicates that systemic disruption of PI3K p110δ function in the TCL1 mouse significantly prevents spontaneous leukemia development, indicating that PI3K p110δ is a critical kinase for CLL disease initiation and expansion. Moreover, inactivation of PI3K p110δ in the microenvironment showed a dose dependent effect in delaying leukemia engraftment. This suggests that PI3K p110δ activity is also critical in the non-B cell compartment for leukemia progression. While our group has focused on the role of PI3K p110δ, we continue to examine the role of PI3K p110γ using the PI3K p110δ and p110γ inhibitor, IPI-145. This study provides solid rationale for ongoing clinical development of IPI-145 in CLL patients. Additionally, as IPI-145 can continue to inhibit the PI3K/AKT pathway in the setting of a C481S BTK mutation, it provides justification for clinical studies of this agent in this subset of patients. Citation Format: Amy J. Johnson, Shuai Dong, Daphne Guinn, Jason A. Dubovsky, Yiming Zhong, Amy Lehman, Jeff Kutok, Jennifer A. Woyach, John C. Byrd. Pharmacologic inhibition with IPI-145 and genetic inhibition of PI3K p110δ antagonizes intrinsic and extrinsic survival signals in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B44.

Atypical presentation and manifestations in X-linked agammaglobulinemia patients with novelBTKmutations

X-linked agammaglobulinemia (XLA) is a rare immunodeficiency caused by defects in the Bruton tyrosine kinase (BTK) gene, characterized by impaired B-cell development, reduced immunoglobulin production, and increased susceptibility to bacterial infections at an early age. Some XLA patients show atypical presentations, with most reports concentrating on the diagnosis at a relatively old age. They presented with infections at late age or with unusual pathogens; however, other atypical manifestations have only rarely been reported.Methods: Description of patients with XLA and novel mutations in BTK who presented with atypical manifestations or developed noninfectious complications.Results: Four patients presented unique manifestations unusual for XLA. The first with Granulomatous Dermatitis, the second with acute demyelinating encephalomyelitis, the third with “Crohn's disease like” localized protein-losing enteropathy, and the last patient with idiopathic thrombocytopenic purpura, which is an unexpected finding in a patient devoid of endogenous immunoglobulins. Mutations in BTK were found in all domains of the gene; 1 resulted in a stop codon and 3 were missense mutations.Conclusions: Early recognition of atypical presentations and manifestations of patients with XLA is crucial for timely initiation of life-saving therapy, which may include anti-bacterial and anti-inflammatory treatments in addition to immunoglobulin.Statement of novelty: In this study we present unique inflammatory and autoimmune phenomenons in XLA patients that were not described previously and are somewhat unexpected. These should alert the immunologist for the possibility of XLA diagnosis.

VIII. Treatment of chronic lymphocytic leukaemia, where are we heading?

VIII. Treatment of chronic lymphocytic leukaemia, where are we heading?

Etiology of Ibrutinib Therapy Discontinuation and Outcomes in Patients With Chronic Lymphocytic Leukemia.

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib is effective in patients with chronic lymphocytic leukemia (CLL). Reasons for discontinuing therapy with this drug and outcomes following discontinuation have not been evaluated outside of clinical trials with relatively short follow-up. To determine features associated with discontinuation of ibrutinib therapy and outcomes. A total of 308 patients participating in 4 sequential trials of ibrutinib at The Ohio State University Comprehensive Cancer Center were included. These clinical trials accrued patients included in this analysis from May 2010 until April 2014, and data were locked in June 2014. Patients were evaluated for time to therapy discontinuation, reasons for discontinuation, and survival following discontinuation. For patients who discontinued therapy because of disease progression, targeted deep sequencing was performed in samples at baseline and time of relapse. With a median follow-up of 20 months, 232 patients remained on therapy, 31 had discontinued because of disease progression, and 45 had discontinued for other reasons. Disease progression includes Richter's transformation (RT) or progressive CLL. Richter's transformation appeared to occur early and CLL progressions later (cumulative incidence at 12 months, 4.5% [95% CI, 2.0%-7.0%] and 0.3% [95% CI, 0%-1.0%], respectively). Median survival following RT was 3.5 months (95% CI, 0.3-6.0 months) and 17.6 months (95% CI, 4.7 months-"not reached") following CLL progression. Sequencing on peripheral blood from 8 patients with RT revealed 2 with mutations in BTK, and a lymph node sample showed no mutations in BTK or PLCG2. Deep sequencing on 11 patients with CLL progression revealed BTK or PLCG2 mutations in all. These mutations were not identified before treatment in any patient. This single-institution experience with ibrutinib confirms it to be an effective therapy and identifies, for the first time, baseline factors associated with ibrutinib therapy discontinuation. Outcomes data show poor prognosis after discontinuation, especially for those patients with RT. Finally, sequencing data confirm initial reports associating mutations in BTK and PLCG2 with progression and clearly show that CLL progressions are associated with these mutations, while RT is likely not. clinicaltrials.gov Identifiers:NCT01105247, NCT01217749, NCT01589302, and NCT01578707.

Splice-correction strategies for treatment of X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease caused by mutations in the gene coding for Bruton’s tyrosine kinase (BTK). Deficiency of BTK leads to a developmental block in B cell differentiation; hence, the patients essentially lack antibody-producing plasma cells and are susceptible to various infections. A substantial portion of the mutations in BTK results in splicing defects, consequently preventing the formation of protein-coding mRNA. Antisense oligonucleotides (ASOs) are therapeutic compounds that have the ability to modulate pre-mRNA splicing and alter gene expression. The potential of ASOs has been exploited for a few severe diseases, both in pre-clinical and clinical studies. Recently, advances have also been made in using ASOs as a personalized therapy for XLA. Splice-correction of BTK has been shown to be feasible for different mutations in vitro, and a recent proof-of-concept study demonstrated the feasibility of correcting splicing and restoring BTK both ex vivo and in vivo in a humanized bacterial artificial chromosome (BAC)-transgenic mouse model. This review summarizes the advances in splice correction, as a personalized medicine for XLA, and outlines the promises and challenges of using this technology as a curative long-term treatment option.

Mutations in Bruton’s tyrosine kinase impair IgA responses

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency caused by mutations in Bruton's tyrosine kinase (BTK), and is characterized by markedly decreased numbers of blood B cells and an absence of all immunoglobulin isotypes. We performed whole exome sequencing in a male pediatric patient with dysgammaglobulinemia with IgA deficiency. Genetic analysis revealed a BTK missense mutation (Thr316Ala). To investigate whether a BTK mutation underlay this antibody deficiency with marked decrease of IgA in this patient, we performed functional analyses of B cells and phagocytes, and molecular analyses of somatic hypermutation and class switch recombination. The BTK missense mutation resulted in B cells with reduced BTK and high IgM expression. Equal proportions of CD19(low) and CD19(normal) fractions were observed, and both included naïve and memory B cells. Calcium influx and phospholipase Cγ2 phosphorylation upon IgM stimulation were marginally impaired in CD19(low), but not in CD19(+) B cells. Similar to XLA patients, IgA transcripts showed low SHM levels, whereas IgG transcripts were hardly affected. Our analyses suggest that the BTK mutation likely underlies the disease in this case, and that hypomorphic BTK mutations can result in normal circulating B cell numbers, but specifically impair IgA responses.

Cell-cycle reprogramming for PI3K inhibition overrides a relapse-specific C481S BTK mutation revealed by longitudinal functional genomics in mantle cell lymphoma.

Despite the unprecedented clinical activity of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib in mantle cell lymphoma (MCL), acquired resistance is common. By longitudinal integrative whole-exome and whole-transcriptome sequencing and targeted sequencing, we identified the first relapse-specific C481S mutation at the ibrutinib binding site of BTK in MCL cells at progression following a durable response. This mutation enhanced BTK and AKT activation and tissue-specific proliferation of resistant MCL cells driven by CDK4 activation. It was absent, however, in patients with primary resistance or progression following transient response to ibrutinib, suggesting alternative mechanisms of resistance. Through synergistic induction of PIK3IP1 and inhibition of PI3K-AKT activation, prolonged early G1 arrest induced by PD 0332991 (palbociclib) inhibition of CDK4 sensitized resistant lymphoma cells to ibrutinib killing when BTK was unmutated, and to PI3K inhibitors independent of C481S mutation. These data identify a genomic basis for acquired ibrutinib resistance in MCL and suggest a strategy to override both primary and acquired ibrutinib resistance. We have discovered the first relapse-specific BTK mutation in patients with MCL with acquired resistance, but not primary resistance, to ibrutinib, and demonstrated a rationale for targeting the proliferative resistant MCL cells by inhibiting CDK4 and the cell cycle in combination with ibrutinib in the presence of BTK(WT) or a PI3K inhibitor independent of BTK mutation. As drug resistance remains a major challenge and CDK4 and PI3K are dysregulated at a high frequency in human cancers, targeting CDK4 in genome-based combination therapy represents a novel approach to lymphoma and cancer therapy. Cancer Discov; 4(9); 1022-35. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 973.

Applying T-cell receptor excision circles and immunoglobulin κ-deleting recombination excision circles to patients with primary immunodeficiency diseases

Background. Biomarkers of T-cell receptor excision circles (TRECs) and immunoglobulin κ-deleting recombination excision circles (KRECs) reflect naïve T and B cell emigrants. This study assessed the biomarkers in patients with primary immunodeficiency diseases (PIDs) to determine the lymphocyte output disturbance and the correlation to lymphocytes.Methods. A standard plasmid was constructed to calculate TRECs and KRECs in 250 ng genomic DNA from whole blood of PIDs patients. These were correlated to naïve and memory lymphocytes for further classification and adequate treatment.Results. In 69 studied patients, the low TRECs mainly included those with severe combined T and B immunodeficiency (SCID, 7/8), combined immunodeficiency (CID, 4/4), and common variable immunodeficiency (CVID, 6/7). The diminished KRECs was in SCID (4/8), CID (4/4), CVID (7/7), Bruton's tyrosine kinase mutation (Btk, 3/4), anti-B cell deletion (by anti-CD20 antibody in 1), and Behçet syndrome under steroid treatment (1). The TRECs and KRECs positively correlated to absolute naïve T (CD4 + CD45RA+) and naïve B (CD19 + CD27−), and to memory B (CD19 + CD27+) numbers, respectively.Conclusion. This study validates that low TRECs and KRECs values reflect low naïve T and B lymphocytes in ‘combined immunodeficiencies’ and in some CVID patients with the potential to develop the CID phenotype.