Therapeutic targeting of Bruton tyrosine kinase (BTK) with ibrutinib in chronic lymphocytic leukemia has led to a paradigm shift in therapy, and relapse has been uncommon with current follow-up. Acquired mutations in BTK and PLCG2 can cause relapse, but data regarding the prevalence and natural history of these mutations are limited. Patients and MethodsPatients accrued to four sequential studies of ibrutinib were included in these analyses. Deep sequencing for BTK and PLCG2 was performed retrospectively on patients who experienced relapse and prospectively on a screening population. ResultsWith a median follow-up time of 3.4 years, the estimated cumulative incidence of progression at 4 years is 19% (95% CI, 14% to 24%). Baseline karyotypic complexity, presence of del(17)(p13.1), and age less than 65 years were risk factors for progression. Among patients who experienced relapse, acquired mutations of BTK or PLCG2 were found in 85% (95% CI, 71% to 94%), and these mutations were detected an estimated median of 9.3 months (95% CI, 7.6 to 11.7 months) before relapse. Of a group of 112 patients examined prospectively, eight patients have experienced relapse, and all of these patients had acquired resistance mutations before relapse. A resistance mutation was detected in an additional eight patients who have not yet met criteria for clinical relapse. ConclusionRelapse of chronic lymphocytic leukemia after ibrutinib is an issue of increasing clinical significance. We show that mutations in BTK and PLCG2 appear early and have the potential to be used as a biomarker for future relapse, suggesting an opportunity for intervention.

Key Points PI3K p110δ/γ inhibitor IPI-145 abrogates prosurvival signals and induces apoptosis in CLL cells. IPI-145 overcomes BTK C481S mutation conferring ibrutinib resistance.

• Selinexor exhibits synergy with ibrutinib in CLL.• Selinexor is effective in vitro in ibrutinib-resistant CLL.Despite the therapeutic efficacy of ibrutinib in chronic lymphocytic leukemia (CLL), complete responses are infrequent, and acquired resistance to Bruton agammaglobulinemia tyrosine kinase (BTK) inhibition is being observed in an increasing number of patients. Combination regimens that increase frequency of complete remissions, accelerate time to remission, and overcome single agent resistance are of considerable interest. We previously showed that the XPO1 inhibitor selinexor is proapoptotic in CLL cells and disrupts B-cell receptor signaling via BTK depletion. Herein we show the combination of selinexor and ibrutinib elicits a synergistic cytotoxic effect in primary CLL cells and increases overall survival compared with ibrutinib alone in a mouse model of CLL. Selinexor is effective in cells isolated from patients with prolonged lymphocytosis following ibrutinib therapy. Finally, selinexor is effective in ibrutinib-refractory mice and in a cell line harboring the BTK C481S mutation. This is the first report describing the combined activity of ibrutinib and selinexor in CLL, which represents a new treatment paradigm and warrants further evaluation in clinical trials of CLL patients including those with acquired ibrutinib resistance. (Blood. 2015;125(20):3128-3132) IntroductionChronic lymphocytic leukemia (CLL) is a lymphoid malignancy of clonal B cells that exhibit aberrant activation of the B-cell receptor (BCR) signaling pathway. A critical component of this pathway is Bruton agammaglobulinemia tyrosine kinase (BTK), a nonreceptor tyrosine kinase expressed predominantly in B lymphocytes.3 Ibrutinib, which irreversibly binds and inhibits BTK activity, has shown promising results in CLL, mantle cell lymphoma, and a subset of diffuse large B-cell lymphoma driven by BCR signaling.4-6 Despite encouraging results, complete responses are infrequent.7 Additionally, acquired resistance to ibrutinib represents an important clinical challenge wherein no standard treatment approach currently exists. Mechanisms of ibrutinib resistance were elucidated by our group and others and involve mutations at the C481S site of BTK or in the immediate downstream target, PLCg2. 1,2,8 Exportin-1 (CRM1/XPO1) is the sole nuclear exporter of tumor suppressor proteins such as p53, inhibitory nuclear factor-kB, and FOXO3a.9,10 Selective inhibitors of nuclear export (SINEs) inhibit XPO1 and restore subcellular localization of dysregulated molecules. Our previous published work showed XPO1 is a therapeutic target for CLL 11 and has facilitated translation of selinexor, a SINE, to a phase 1 clinical trial (#NCT01607892), where antitumor activity has been observed in lymphoma, 12 CLL, 12 multiple myeloma, 13 and acute myeloid leukemia.14 We recently showed that selinexor inhibits activation of downstream BCR targets such as extracellular signal-regulated kinase and protein kinase B and suppresses BTK gene expression. 15 Based on t...

The clinical success of ibrutinib validates Bruton tyrosine kinase (BTK) inhibition as an effective strategy for treating hematologic malignancies, including chronic lymphocytic leukemia (CLL). Despite ibrutinib's ability to produce durable remissions in patients, acquired resistance can develop, mostly commonly by mutation of C481 of BTK in the ibrutinib binding site. Here, we characterize a novel BTK inhibitor, GDC-0853, to evaluate its preclinical efficacy in ibrutinib-naive and ibrutinib-resistant CLL. GDC-0853 is unique among reported BTK inhibitors in that it does not rely upon covalent reaction with C481 to stabilize its occupancy within BTK's adenosine triphosphate binding site. As with ibrutinib, GDC-0853 potently reduces B-cell receptor signaling, viability, NF-κB-dependent transcription, activation, and migration in treatment naïve CLL cells. We found that GDC-0853 also inhibits the most commonly reported ibrutinib-resistant BTK mutant (C481S) both in a biochemical enzyme activity assay and in a stably transfected 293T cell line and maintains cytotoxicity against patient CLL cells harboring C481S BTK mutations. Additionally, GDC-0853 does not inhibit endothelial growth factor receptor or ITK, 2 alternative targets of ibrutinib that are likely responsible for some adverse events and may reduce the efficacy of ibrutinib-antibody combinations, respectively. Our results using GDC-0853 indicate that noncovalent, selective BTK inhibition may be effective in CLL either as monotherapy or in combination with therapeutic antibodies, especially among the emerging population of patients with acquired resistance to ibrutinib therapy.

Objectives: We evaluated the bioavailability of Cd in 86 components of 57 jewelry items found to contain high levels of Cd (> 10,000 ppm) by X-ray fluorescence (XRF), using extractions that simulate mouthing or swallowing of jewelry items.Methods: We screened jewelry for Cd content by XRF. Bioavailability was measured in two ways. Items were placed in saline solution at 37°C for 6 hr to simulate exposures from mouthing of jewelry items. Items were placed in dilute hydrochloric acid (HCl) at 37°C for 6–96 hr, simulating the worst-case scenario of a child swallowing a jewelry item. Damaged pieces of selected samples were also extracted by both methods to determine the effect of breaching the outer plating on bioavailability. Total Cd content of all items was determined by atomic absorption.Results: The 6-hr saline extraction yielded as much as 2,200 µg Cd, and 24-hr dilute HCl extraction yielded a maximum of > 20,000 µg Cd. Leaching of Cd in dilute HCl increased linearly over 6–96 hr, indicating potential for increasing harm the longer an item remains in the stomach. Damage to jewelry by breaching the outer plating generally, but not always, increased Cd release. Bioavailability did not correlate directly with Cd content.Conclusions: These results indicate the potential for dangerous Cd exposures to children who wear, mouth, or accidentally swallow high-Cd jewelry items.