First-in-Human Study of the Reversible BTK Inhibitor Nemtabrutinib in Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia and B-Cell Non-Hodgkin Lymphoma.

Nemtabrutinib is an orally bioavailable, reversible inhibitor of Bruton tyrosine kinase (BTK) and C481S mutant BTK. We evaluated the safety, pharmacology, and antitumor activity of nemtabrutinib in relapsed/refractory hematologic malignancies. Forty-eight patients with chronic lymphocytic leukemia (CLL), B-cell non-Hodgkin lymphoma (NHL), or Waldenström macroglobulinemia (WM), relapsed/refractory after ≥2 prior therapies were enrolled in the open-label, single-arm, phase I MK-1026-001 study (NCT03162536) to receive nemtabrutinib 5 to 75 mg once daily in 28-day cycles. Dose finding progressed using a 3 + 3 dose escalation design. Primary endpoints were safety and the recommended phase II dose (RP2D). Among 47 treated patients, 29 had CLL, 17 had NHL, and 1 had WM. Grade ≥3 treatment-emergent adverse events occurred in 37 (89%), most commonly neutropenia (11; 23.4%), febrile neutropenia (7; 14.9%), and pneumonia (7; 14.9%). The RP2D was 65 mg daily. An overall response rate of 75% was observed in patients with CLL at 65 mg daily. SIGNIFICANCE: This first-in-human phase I study demonstrates the safety and preliminary efficacy of nemtabrutinib in patients with relapsed/refractory B-cell malignancies. These data support further exploration of nemtabrutinib in larger clinical studies. This article is featured in Selected Articles from This Issue, p. 5.

Dual TCR-Expressing T Cells in Cancer: How Single-Cell Technologies Enable New Investigation.

TCR diversity measures are often used to understand the immune response in cancer. Traditional measures of diversity rely on bulk RNA sequencing (RNAseq) of the ß-chain variable regions. However, the full αß TCR repertoire is a combination of both the α- and ß-chains, which are encoded by separate genes. In contrast with bulk RNAseq, single-cell RNAseq (scRNAseq) allows paired chain analyses, yielding a more accurate measure of the repertoire. Interestingly, ∼30% of mature peripheral T cells express multiple TCR alleles (e.g., two α-chains) and may exhibit dual Ag specificity. scRNAseq has become increasingly common, and data from both human and animal studies are publicly available. However, routine workflows discard secondary TCR alleles and focus on a single TCR clone per cell. This perspectives piece emphasizes why this may not be good practice and highlights unanswered questions in the field of T cell dual specificity.

Preclinical evaluation of combination nemtabrutinib and venetoclax in chronic lymphocytic leukemia.

Inhibitors of B cell receptor (BCR) signaling such as the Bruton's tyrosine kinase (BTK) inhibitors are effective therapeutics for chronic lymphocytic leukemia (CLL). The first-in-class covalent BTK inhibitor, ibrutinib, produces durable responses in most CLL patients; however, complete responses are only observed in a minority of patients. B cell lymphoma 2 (BCL2), an anti-apoptotic protein that contributes to CLL cell survival, has also been investigated as a therapeutic target. The BCL2 inhibitor venetoclax is effective in patients with CLL and can produce undetectable minimal residual disease, allowing discontinuation of therapy. In combination, ibrutinib and venetoclax have shown preclinical synergy and clinical efficacy. Nemtabrutinib is a next generation, reversible inhibitor of BTK that potently inhibits BCR signaling in treatment-naïve and ibrutinib-refractory CLL cells ex vivo. The clinical efficacy of combining BTK inhibitors with BCL2 inhibitors motivated us to evaluate the novel combination of nemtabrutinib and venetoclax. In vitro studies show that nemtabrutinib and venetoclax are not antagonistic to each other. In an adoptive transfer CLL mouse model, mice treated with nemtabrutinib and venetoclax had prolonged survival compared to mice treated with ibrutinib and venetoclax. Our preclinical studies further validate the combination of BTK inhibitors with venetoclax and justify further investigation of combining nemtabrutinib with venetoclax in CLL.

Intentional Modulation of Ibrutinib Pharmacokinetics through CYP3A Inhibition.

Ibrutinib (Imbruvica; PCI-32765) is an orally administered inhibitor of Bruton's tyrosine kinase that has transformed the treatment of B-cell malignancies. However, ibrutinib has very low oral bioavailability that contributes to significant variability in systemic exposure between patients, and this has the potential to affect both efficacy and toxicity. We hypothesized that the oral bioavailability of ibrutinib is limited by CYP3A isoform-mediated metabolism, and that this pathway can be inhibited to improve the pharmacokinetic properties of ibrutinib. Pharmacokinetic studies were performed in wild-type mice and mice genetically engineered to lack all CYP3A isoforms [CYP3A(-/-)] that received ibrutinib alone or in combination with CYP3A inhibitors cobicistat or ketoconazole. Computational modeling was performed to derive doses of ibrutinib that, when given after a CYP3A inhibitor, results in therapeutically-relevant drug levels. Deficiency of CYP3A in mice was associated with a ~10-fold increase in the area under the curve of ibrutinib. This result could be phenocopied by administration of cobicistat before ibrutinib in wild-type mice, but cobicistat did not influence levels of ibrutinib in CYP3A(-/-) mice. Population pharmacokinetic and prospectively validated physiologically-based pharmacokinetic models established preclinical and clinical doses of ibrutinib that could be given safely in combination with cobicistat without negatively affecting anti-leukemic properties. These findings signify a dominant role for CYP3A-mediated metabolism in the elimination of ibrutinib, and suggest a role for pharmacological inhibitors of this pathway to intentionally modulate the plasma levels and improve the therapeutic use of this clinically important agent.

Targeting phosphatidylinositol 3 kinase-ß and -δ for Bruton tyrosine kinase resistance in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma; 40% of patients relapse after a complete response or are refractory to therapy. To survive, the activated B-cell (ABC) subtype of DLBCL relies upon B-cell receptor signaling, which can be modulated by the activity of Bruton tyrosine kinase (BTK). Targeting BTK with ibrutinib, an inhibitor, provides a therapeutic approach for this subtype of DLBCL. However, non-Hodgkin lymphoma is often resistant to ibrutinib or acquires resistance soon after exposure. We explored how this resistance develops. We generated 3 isogenic ibrutinib-resistant DLBCL cell lines and investigated the deregulated pathways known to be associated with tumorigenic properties. Reduced levels of BTK and enhanced phosphatidylinositol 3-kinase (PI3K)/AKT signaling were hallmarks of these ibrutinib-resistant cells. Upregulation of PI3K-ß expression was demonstrated to drive resistance in ibrutinib-resistant cells, and resistance was reversed by the blocking activity of PI3K-ß/δ. Treatment with the selective PI3K-ß/δ dual inhibitor KA2237 reduced both tumorigenic properties and survival-based PI3K/AKT/mTOR signaling of these ibrutinib-resistant cells. In addition, combining KA2237 with currently available chemotherapeutic agents synergistically inhibited metabolic growth. This study elucidates the compensatory upregulated PI3K/AKT axis that emerges in ibrutinib-resistant cells.

Ibrutinib reprograms the glucocorticoid receptor in chronic lymphocytic leukemia cells.

Glucocorticoid (GC) receptor (GR) phosphorylation and signature genes were studied in chronic lymphocytic leukemia (CLL) cells to help place GCs within modern treatment algorithms. In contrast to normal B and T cells, transcription of GC-regulated genes was not rhythmic and the synthetic GC dexamethasone (DEX) could not inhibit toll-like receptor (TLR)-responses in CLL cells. This intrinsic GC-resistance was associated with aberrant GR-phosphorylation on activating Ser211 and inhibitory Ser226 sites. Ibrutinib increased transcription of the GR-signature gene GILZ in circulating CLL cells along with GR(pS211)/GR(pS226) ratios and lytic sensitivity to DEX that were not reversed by the competitive antagonist mifepristone in vitro. However, ibrutinib could not improve GR-responses in circulating CLL cells activated with IL2 and TLR7/8 agonists to mimic conditions in pseudofollicle microenvironments. Addition of the janus kinase inhibitor ruxolitinib to block ibrutinib-insensitive signals increased GILZ transcription in pseudofollicle conditions in vitro and in a clinical trial (NCT02912754), and also increased GR(S211)/GR(S226) ratios and DEX-mediated killing in patient samples in vitro. These observations suggest that intrinsic resistance to endogenous GCs is characteristic of CLL cells and ibrutinib may help increase the therapeutic activity of GCs by non-canonical activation of GR.

The BTK Inhibitor ARQ 531 Targets Ibrutinib-Resistant CLL and Richter Transformation.

Targeted inhibition of Bruton tyrosine kinase (BTK) with the irreversible inhibitor ibrutinib has improved outcomes for patients with hematologic malignancies, including chronic lymphocytic leukemia (CLL). Here, we describe preclinical investigations of ARQ 531, a potent, reversible inhibitor of BTK with additional activity against Src family kinases and kinases related to ERK signaling. We hypothesized that targeting additional kinases would improve global inhibition of signaling pathways, producing more robust responses. In vitro treatment of patient CLL cells with ARQ 531 decreases BTK-mediated functions including B-cell receptor (BCR) signaling, viability, migration, CD40 and CD86 expression, and NF-κB gene transcription. In vivo, ARQ 531 was found to increase survival over ibrutinib in a murine Eµ-TCL1 engraftment model of CLL and a murine Eµ-MYC/TCL1 engraftment model resembling Richter transformation. Additionally, ARQ 531 inhibits CLL cell survival and suppresses BCR-mediated activation of C481S BTK and PLCγ2 mutants, which facilitate clinical resistance to ibrutinib.Significance: This study characterizes a rationally designed kinase inhibitor with efficacy in models recapitulating the most common mechanisms of acquired resistance to ibrutinib. Reversible BTK inhibition is a promising strategy to combat progressive CLL, and multikinase inhibition demonstrates superior efficacy to targeted ibrutinib therapy in the setting of Richter transformation. Cancer Discov; 8(10); 1300-15. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1195.

Noncovalent inhibition of C481S Bruton tyrosine kinase by GDC-0853: a new treatment strategy for ibrutinib-resistant CLL.

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.