Targeting Bruton's Tyrosine Kinase Research Articles

A Phase 1 Study of the Combination of Acalabrutinib and AZD9150 in Patients with Relapsed/Refractory Diffuse Large B-Cell Lymphoma

A Phase 1 Study of the Combination of Acalabrutinib and AZD9150 in Patients with Relapsed/Refractory Diffuse Large B-Cell Lymphoma

Nx-5948, a Selective Degrader of BTK with Activity in Preclinical Models of Hematologic and Brain Malignancies

Bruton's tyrosine kinase (BTK) plays a key role in cell survival in B cell malignancies, and covalent inhibitors of BTK, such as ibrutinib and acalabrutinib, have proven efficacious in chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and Waldenstrom's macroglobulinemia (WM). BTK inhibitors have also demonstrated clinical activity in small trials of patients with relapsed/refractory primary central nervous system lymphoma. The long-term efficacy of BTK inhibitors is limited by the emergence of resistance mutations, most commonly at C481 of BTK. These mutations preclude formation of a covalent bond with BTK and lead to diminished efficacy and disease progression. Several noncovalent BTK inhibitors, which do not require covalent binding to C481, are currently being investigated in clinical trials as potential therapies for patients with relapsed and refractory disease. However, other mutations have been shown to decrease the in vitro activity of these non-covalent BTK inhibitors, suggesting that mutations may ultimately limit the effectiveness of these compounds as well.Small molecule-induced protein degradation offers a unique approach to target BTK for the treatment of B-cell malignancies. Chimeric Targeting Molecules (CTMs) catalyze ubiquitylation and proteasomal degradation of target proteins and are comprised of a target binding element (“hook”), a linker, and ubiquitin ligase binding element (“harness”). NX-5948 is a CTM that contains a BTK hook linked to a cereblon (CRBN) harness. While some CRBN-binding drugs, such as lenalidomide and pomalidomide, promote the degradation of neo-substrate proteins, such as the transcription factors Aiolos and Ikaros, NX-5948 has been engineered to avoid Aiolos and Ikaros degradation and therefore does not possess IMiD activity.Preclinical studies have shown that NX-5948 catalyzes the degradation of 50% of cellular BTK (DC 50) at < 1 nM concentrations in lymphoma cell lines and peripheral blood mononuclear cells (PBMCs). NX-5948 impairs viability in the BTK-dependent ABC-DLBCL cell line, TMD8 (EC 50: < 10 nM after 72 hours). Importantly, NX-5948 induces degradation of the mutated BTK-C481S in cells and inhibits proliferation of BTK-C481S mutant TMD8 cells more effectively than ibrutinib (NX-5948 EC 50 values of < 10 nM versus > 1 μM for ibrutinib). NX-5948 also catalyzes selective degradation of BTK. In an unbiased proteomics experiment, NX-5948 treatment of TMD8 cells demonstrates robust BTK degradation without significant downregulation of other off-target proteins.Oral administration of NX-5948 in mice leads to dose-proportional exposure in plasma and BTK degradation to <10% of baseline levels in circulating and splenic B cells. In a TMD8 xenograft model in mice containing the BTK-C481S mutation, daily oral administration of NX-5948 resulted in superior tumor growth inhibition (TGI) as compared to ibrutinib. Following a single oral dose of NX-5948 in cynomolgus monkeys, BTK degradation to <10% of baseline levels is observed in the circulating B cells.In addition, NX-5948 penetrates the central nervous system (CNS) and demonstrates activity in a model of brain malignancies. Following oral dosing of NX-5948 to mice, NX-5948 was detectable in the cerebrospinal fluid at a comparable level to the unbound concentration of NX-5948 in mouse plasma. In an intracranial TMD8 xenograft model, treatment with NX-5948 resulted in degradation of BTK in intracranial TMD8 cells, decreased intracranial tumor burden and improved survival relative to treatment with vehicle.The potent BTK degradation activity of NX-5948 and the ability of this molecule to penetrate the CNS supports its development for the treatment of B-cell malignancies, including CNS lymphoma. DisclosuresRobbins: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Noviski: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rountree: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tan: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Brathaban: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ingallinera: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Karr: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Kelly: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Konst: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ma: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tenn-McClellan: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. McKinnell: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Perez: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Guiducci: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hansen: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sands: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

Myricetin exhibit selective anti-lymphoma activity by targeting BTK and is effective via oral administration in vivo

BackgroundMyricetin (MYR) is a polyhydroxy flavone originally isolated from Myrica rubra, and is widely distributed in a variety of medicinal plants and delicious food. MYR has been proven to have inhibitory effects against various types of cancer. However, the exact role of MYR in lymphoma development is still unclear. MethodsIn vitro, the MTT assay was performed to evaluate the activity of human diffuse large B lymphoma cell TMD-8 and other tumor cells. Homogeneous time-resolved fluorescence (HTRF) and molecular docking were used to detect the target of MYR inhibiting TMD-8 cells. In addition, flow cytometry, Annexin V-FITC/PI assays, Hoechst 33258, and mondansylcadaverine (MDC) fluorescent standing were used to detect the cell cycle, apoptosis, and autophagy, respectively. Moreover, Western blot analysis was conducted to analyze related signaling pathways. In TMD-8 cell xenotransplanted mice, immunohistochemistry, histopathology, and blood biochemical tests were used to evaluate the effectiveness and safety of oral administration of MYR. ResultsHere, we found that MYR is more sensitive to TMD-8 cells than other tumor cells by targeting bruton tyrosine kinase (BTK). BTK is an attractive target for the treatment of B-cell malignancies. The HTRF assay showed that MYR inhibited BTK kinase with an IC50 of 1.82 μM. Furthermore, the HTRF assay and Western blot analysis demonstrated that MYR could bind to key residues (Ala478, Leu408, Thr474) in the BTK active pocket, inhibit the autophosphorylation on tyrosine 223, and block BTK/ERK and BTK/AKT signal transduction cascades (including downstream substrates GSK-3β, IKK, STAT3, and NF-κb). The results of cell cycle, apoptosis, and autophagy showed that MYR could induce G1/G0 cycle arrest by regulating cyclinB1/D1 expression, induce apoptosis by increasing the Bax/Bcl-2 ratio, and trigger autophagy by inhibiting mTOR activation. In vivo, oral administration of MYR significantly inhibited the growth of TMD-8 xenograft tumora without toxic side effects. Furthermore, Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis showed that MYR could inhibit proliferation and induce apoptosis of tissue lymphoma cells. ConclusionTaken together, MYR is an oral available natural BTK inhibitor that effectively inhibits the growth of lymphoma TMD-8 cells both in vitro and in vivo. In addition, our findings support that the use of MYR is a novel and promising therapeutic strategy for the treatment of lymphoma.

Richter transformation of chronic lymphocytic leukaemia: a British Society for Haematology Good Practice Paper.

Richter transformation of chronic lymphocytic leukaemia: a British Society for Haematology Good Practice Paper.

Targeting Bruton's tyrosine kinase in primary central nervous system lymphoma.

We review the preclinical and clinical experience with first and subsequent generation Bruton's tyrosine kinase inhibitors in B-cell lymphoproliferative diseases, highlighting the rationale for their clinical use in primary central nervous system diffuse large B-cell lymphoma (PCNSL). Growing knowledge on the molecular and genetic profile of PCNSL has provided the basis for new drug development targeting aberrantly activated oncogenic signal transduction pathways. PCNSL exhibits frequent genetic alterations of components of the B-cell and Toll-like receptor signalling pathways. On the basis of these discoveries and the limited efficacy obtained with chemotherapy in refractory and relapsed PCNSL, activity of new targeted agents, such as Bruton's tyrosine kinase inhibitors, has been explored with promising results. Innovative therapeutic strategies, applied in first line, have contributed to improved outcomes in patients with PCNSL, making this disease potentially curable in young and fit patients. However, response to induction therapies remains suboptimal and the best consolidative therapy has yet to be defined. In this regard, given the activity of Bruton's tyrosine kinase inhibitors in the refractory and relapsed PCNSL setting, these agents are currently being explored as part of combination regimens for induction therapy of newly diagnosed PCNSL.

Hairy cell leukemia: a brief update on current knowledge and treatment prospects.

This article provides a brief update on the recommended diagnosis and treatment strategies for patients with the classic form of hairy cell leukemia (HCL) and HCL variant (HCLv). HCL is a chronic B-cell malignancy with multiple treatment options. In recent years, many novel drugs have been assessed for HCL treatment with promising results. The investigated nonchemotherapy options include moxetumomab pasudotox, which targets CD22; vemurafenib or dabrafenib, which target the BRAFV600E protein; trametinib, which targets mitogen-activated protein kinase enzyme; and ibrutinib, which targets Bruton tyrosine kinase. Purine analogs significantly improve survival in patients with HCL. However, patients often relapse, require multiple treatments, and may become refractory. The introduction of novel agents has expanded the spectrum of therapy possibilities in those patients. In the coming years, they will assist standard therapy for patients with HCL who may currently have suboptimal results.

Bruton's Tyrosine Kinase and Its Isoforms in Cancer.

Bruton’s tyrosine kinase (BTK) is a soluble tyrosine kinase with central roles in the development, maturation, and signaling of B cells. BTK has been found to regulate cell proliferation, survival, and migration in various B-cell malignancies. Targeting BTK with recently developed BTK inhibitors has been approved by the Food and Drug Administration (FDA) for the treatment of several hematological malignancies and has transformed the treatment of several B-cell malignancies. The roles that BTK plays in B cells have been appreciated for some time. Recent studies have established that BTK is expressed and plays pro-tumorigenic roles in several epithelial cancers. In this review, we focus on novel isoforms of the BTK protein expressed in epithelial cancers. We review recent work on the expression, function, and signaling of these isoforms and their value as potential therapeutic targets in epithelial tumors.

Dissection of Clonal Evolution By Temporal Mutation Profiling in Chronic Lymphocytic Leukemia Patients Treated with Ibrutinib

Introduction: The irreversible Bruton tyrosine kinase (BTK) inhibitor ibrutinib is changing the paradigm of the treatment of chronic lymphocytic leukemia (CLL) with remarkable outcomes in first line as well as in relapsed CLL, including high risk patients with TP53 aberration. While the majority of patients demonstrate durable responses, with a median follow up of 3 years, approximately 18% of them develop resistance and relapse. Mutations in the BTK and PLCG2 genes emerged as predominant mechanisms conferring secondary ibrutinib resistance with BTK and/or PLCG2 mutations detected in the majority of resistant patients, often even 10 months before the clinical relapse. Given the very poor outcome of patients discontinuing ibrutinib, comprehensive understanding of the mechanisms of ibrutinib resistance and changes in the clonal architecture induced by the selective pressure of the drug are of major clinical importance. To dissect the clonal evolution in the context of all relevant mutation targets in CLL, we performed a temporal mutation profiling of 31 genes in paired pre- and post-treatment samples of patients receiving ibrutinib therapy.Methods: Sequential samples from 18 patients treated with single-agent ibrutinib were included in this study. Pre-treatment peripheral blood samples were available in all patients, with post-treatment samples at 18 months for 6 patients, at 12 months for 7 patients, at 8 months for 3 patients and at 3 months for 2 patients, with a median follow up of 12 months. Targeted deep NGS analysis of 31 recurrently mutated target genes, including ATM, BCOR, BIRC3, BRAF, BTK, CHD2, DDX3X, EGR2, EIF2A, EP300, FBXW7, HIST1H1E, IGLL5, KLHL6, KMT2D, LRP1B, MAPK1, MED12, MGA, MYD88, NFKBIE, NOTCH1, PLCG2, POT1, RIPK1, RPS15, SAMHD1, SF3B1, TP53, XPO1 and ZMYM3 was performed using the TruSeq Custom Amplicon approach (Illumina) on genomic DNA specimens extracted from peripheral blood mononuclear cells. Variant calling was performed using the VariantStudio3.0 application (Illumina) and only variants supported with more than 100 mutant reads were considered. Our cohort represented a heavily pretreated patient group with a median of 2.5 (range: 1-5) lines of prior therapies.Results: The ultra-deep NGS analysis revealed a total of 473 somatic mutations in the 18 paired samples with a median allelic depth of 25.550x across the 31 genes analyzed. The post-treatment samples carried a considerably higher number of mutations compared to the pre-treatment samples (114 vs 359 mutations) with more than half of the variants representing subclonal alterations with variant allele frequencies (VAF) of <10%. The most frequently mutated genes at baseline included TP53 (50%) , NOTCH1 (50%) and POT1 (28%) with similarly high mutation frequencies in the post-treatment samples. MYD88, BIRC3 and SF3B1 mutations were enriched in the post-ibrutinib samples with mutation frequencies of 11% vs 33%, 11% vs 39% and 11% vs 50%, respectively. Interestingly, XPO1 mutations emerged only in the post-treatment samples of five (28%) patients. As for the BTK and PLCG2 mutations, none of the pre-treatment samples carried a mutation in either of these genes, however, we detected emerging subclonal BTK and PLCG2 mutations (with VAFs between 1.5% and 11%) in 28% (5/18) and 33% (6/18) of the post-treatment samples, respectively. A subset of cases harbored multiple mutations in both genes with some of these representing novel BTK and PLCG2 mutations affecting previously unreported amino acid residues. Scrutiny of the clonal evolution revealed that all individual cases were characterized by almost unique combinations of mutations as well as patterns of emerging and disappearing mutant subclones upon the selective pressure of ibrutinib.Conclusions: The ultra-deep NGS analysis performed on paired samples of CLL patients treated with ibrutinib provided a significant insight into the mutational repertoire as well as subclonal dynamics of the leukemic compartment. Furthermore, it has revealed a profound subclonal heterogeneity that seems to be further enhanced by ibrutinib therapy, as demonstrated by a higher frequency of subclonal variants in the post-treatment samples. The clinical significance and the potential role of the novel BTK and PLCG2 variants identified in our study will require a longer follow up time, as currently all patients harboring these subclonal variants are still on ibrutinib therapy. DisclosuresNo relevant conflicts of interest to declare.

Preclinical Activity of Novel BET Protein Proteolysis Targeting Chimeras (BETP-PROTACs) and Their Combination with Venetoclax or CDK4/6 Inhibitor Against Human Mantle Cell Lymphoma (MCL) Cells

Among the genetic alterations described in Mantle Cell Lymphoma (MCL) are those involving cyclin D1, deletion of INK4a/ARF, as well as copy number gains of MYC, CDK4 and BCL2 genes. Activated B-cell receptor (BCR) signaling, and the ensuing downstream pro-growth and pro-survival NFkB activity, is also a notable feature of MCL. Collectively, the genetic alterations and the ensuing deregulated signaling and activity of transcription factors, including c-MYC and NFkB, creates the MCL-specific ‘transcriptome’ that promotes growth and survival of MCL cells. Ibrutinib, a covalent inhibitor of Bruton's tyrosine kinase (BTK), exhibits unprecedented single-agent activity in relapsed/refractory MCL, however approximately 40% of patients demonstrate primary resistance and secondary resistance is common, with a poor one-year survival rate of only 22%. Mutations in CARD11/IKBKB/TRAF2/BIRC3/NIK or the C481S mutation in BTK, sustain alternative or classical NFkB signaling despite ibrutinib treatment and confer resistance to ibrutinib in MCL. We hypothesized that the resistance to ibrutinib in MCL cells would be governed by a dysregulated transcriptome dependent on the activity of the chromatin reader BET proteins (BETPs), including BRD4. Therefore, we determined the lethal preclinical activity of BETP PROTACs (proteolysis targeting chimeras) ARV-825, and its pharmacologically superior version ARV-771, that proteasomally degrade BETPs, against the following cellular models of ibrutinib resistance (MCL-IR): in vitro generated cultured MCL Mino-persister/resistant (Mino-P/R) cells (~4-fold resistant compared to parental Mino cells), endogenously resistant cultured Z138 and Maver-1 cells, as well as patient-derived xenograft (PDX) PDX1 and PDX2 cells that had exhibited in vivo resistance to ibrutinib. Protein expression evaluations by RPPA and/or Western analyses demonstrated that MCL-IR cells expressed relatively high levels of p-AKT over AKT, p-S6, p-4EBP1, MYC, BCL2, BCl-xL, XIAP and MCL1. Treatment with BETP-PROTAC dose-dependently (20 to 200 nM) induced significantly more growth inhibition and apoptosis than the BETP inhibitor BETi OTX015 against the cellular models of MCL-IR (p < 0.001), which was associated with marked depletion of BRD4, BRD2, MYC, BCL2, BCL-xL, cyclin D1, CDK4 and pRB (S807/811) levels, as well as of NFkB target gene expressions, including cIAP2, XIAP, cFLIP, TNFAIP3, BCl-xL, IL10, TNFa and BTK. Concomitantly, BETP-PROTAC increased expression of HEXIM1 and p21 levels in the MCL-IR cells. Treatment with the BCL2 inhibitor venetoclax or the CDK4/6 inhibitor abemaciclib dose-dependently induced apoptosis of MCL-IR cell types (p< 0.01). Notably, co-treatment with BETP-PROTAC ARV-771 and venetoclax or abemaciclib synergistically induced apoptosis of Mino-P/R, Maver-1 and PDX1 and PDX2 cells (combination indices < 1.0). Additionally, compared to treatment with vehicle control, treatment of luciferase-transduced PDX2 cells infused by the tail vein and engrafted in immune-depleted NSG mice with ARV-771 (30 mg/kg SQ daily) and venetoclax (15 mg/kg PO daily) for 3 weeks, significantly reduced the MCL burden (at 2 weeks of treatment) and improved survival of the mice. These findings demonstrate that BETP-PROTACs deplete BETPs and important MCL-relevant oncogene levels, including MYC, cyclin D1, CDK4, BCL2, Bcl-xL, as well as NFkB target gene expressions, leading to synergistic lethality in vitro in combination with venetoclax or abemaciclib, as well as exert in vivo activity in combination with venetoclax against PDX model of MCL-IR cells. DisclosuresRaina:4Arvinas, LLC. New Haven, CT: Employment. Qian:4Arvinas, LLC. New Haven, CT: Employment. Crew:Arvinas, Inc: Employment.

RAC2 Links B-Cell Receptor Signaling and Cell Adhesion in Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is an incurable non-Hodgkin B-cell lymphoma. B-cell receptor (BCR) signaling is chronically active in several mature B-cell malignancies including mantle cell lymphoma. Inhibition of the BCR and downstream pathway is highly effective in B-cell neoplasia and can be achieved at the cellular level through BTK inhibition by ibrutinib, for example. Ibrutinib has generated remarkable patients' response in large clinical trials and has been approved by FDA for the treatment of relapsed/refractory MCL. Ibrutinib targets Bruton tyrosine kinase primarily by inhibition of B-cell receptor signaling and cell proliferation, and secondarily by induction of tumor apoptosis. In addition, the drug inhibits the interaction of tumor cells with their microenvironment by disruption of cell adhesion. The effect on cell adhesion appears to be a mechanism shared by many BCR-targeted therapies including inhibitors of LYN (Leuk Res 38, 34, 2014), SYK (Blood, 115, 2578, 2010; Blood 125, 2336, 2015), and PI3K (idelalisib) (NEJM, 370, 997, 2014; Blood, 125, 2306). This class action raised a question regarding how the BCR pathway is connected to the cell adhesion phenotype. We set out to investigate the underlying mechanisms that are largely unknown at the present time.Methods: We used RNA-sequencing to identify pathways that have differential responses to ibrutinib in MCL cell lines that are either intrinsically sensitive or resistant to ibrutinib. We then validate the RNA-Seq findings in sensitive and resistant cell lines via RNA interference and transfection, xenograft mouse models, and human primary MCL samples.Results: We found that sensitivities of six MCL cell lines to ibrutinib correlated well with their cell adhesion phenotype. These include sensitive cell line Jeko-1, intermediate cell lines Mino, REC-1 and JVM2, and resistant lines Maver-1 and Granta 519. RNA-sequencing revealed that BCR signaling and cell adhesion transcriptional programs were coordinately down-regulated by ibrutinib in ibrutinib-sensitive cells, but not in ibrutinib-resistant cells. Among genes that are differentially regulated in the sensitive and resistant cell lines, we identified RAC2, a regulator of cell adhesion this is also involved in BCR signaling. To determine whether RAC2 is relevant, we examined RAC2 expression in primary MCL tumors by immunohistochemical staining (IHC). We found that RAC2 was weakly expressed in 7/10 classic MCL tumors and strongly expressed in 3/3 aggressive MCL. In contrast, in tonsilar lymphoid reactive tissues, RAC2 positivity was restricted only to mantle zone and T-zone of the follicles. For further validation, we found that RAC2 expression, at the protein level, was down-regulated by ibrutinib treatment in sensitive, but not resistant, cell lines. More interestingly, using co-immunoprecipitation, we found that RAC2 is physically associated with BLNK, an adaptor of early BCR signaling pathway, in the sensitive cells but not in the resistant cells. To establish that RAC2 is a regulator of MCL cell adhesion in the context of MCL, we demonstrated that siRNA knock-down of RAC2 impaired cell adhesion while exogenous introduction of RAC2 reversed ibrutinib-induced cell adhesion impairment. In a xenograft mouse model, mice treated with ibrutinib showed slower tumor growth with reduced RAC2 expression by IHC. Lastly, we show that in primary human MCL cells, RAC2 was down-regulated by ibrutinib, and this down-regulation correlated well with cell adhesion impairment following ibrutinib treatment.Conclusions: Our findings suggest the RAC2 mediates the link between BCR signaling and cell adhesion. Ibrutinib inhibits cell adhesion via down-regulation of RAC expression and via disruption of RAC2-BLNK interaction. This study highlights the importance of RAC2 and cell adhesion in MCL pathogenesis and drug development. DisclosuresLu:Portola Pharmacueticals, Inc.: Research Funding. Wang:Portola: Research Funding; Karyopharm: Research Funding.

A Pilot Study of Lower Doses of Ibrutinib in Patients (pts) with Chronic Lymphocytic Leukemia (CLL)

A Pilot Study of Lower Doses of Ibrutinib in Patients (pts) with Chronic Lymphocytic Leukemia (CLL)

Targeting Bruton's Tyrosine Kinase in Inflammatory and Autoimmune Pathologies.

Bruton’s tyrosine kinase (BTK) was discovered due to its importance in B cell development, and it has a critical role in signal transduction downstream of the B cell receptor (BCR). Targeting of BTK with small molecule inhibitors has proven to be efficacious in several B cell malignancies. Interestingly, recent studies reveal increased BTK protein expression in circulating resting B cells of patients with systemic autoimmune disease (AID) compared with healthy controls. Moreover, BTK phosphorylation following BCR stimulation in vitro was enhanced. In addition to its role in BCR signaling, BTK is involved in many other pathways, including pattern recognition, Fc, and chemokine receptor signaling in B cells and myeloid cells. This broad involvement in several immunological pathways provides a rationale for the targeting of BTK in the context of inflammatory and systemic AID. Accordingly, numerous in vitro and in vivo preclinical studies support the potential of BTK targeting in these conditions. Efficacy of BTK inhibitors in various inflammatory and AID has been demonstrated or is currently evaluated in clinical trials. In addition, very recent reports suggest that BTK inhibition may be effective as immunosuppressive therapy to diminish pulmonary hyperinflammation in coronavirus disease 2019 (COVID-19). Here, we review BTK’s function in key signaling pathways in B cells and myeloid cells. Further, we discuss recent advances in targeting BTK in inflammatory and autoimmune pathologies.

Proteolysis Targeting Chimeras for BTK Efficiently Inhibit B-Cell Receptor Signaling and Can Overcome Ibrutinib Resistance in CLL Cells.

Proteolysis targeting chimeras (PROTACs) are small molecules that form ternary complexes between their target and E3 ligase, resulting in ubiquitination and proteasomal degradation of the target protein. Using our own designed Bruton’s tyrosine kinase (BTK) PROTAC compounds, we show herein efficient BTK degradation in chronic lymphocytic leukemia (CLL) cells. The reversible non-covalent compound (NC-1) was the most potent and therefore we focused on this PROTAC to investigate its subsequent effects on the BCR pathway. NC-1 decreased baseline BTK phosphorylation as well as activation of BTK and other signaling molecules downstream of the BCR pathway, following IgM engagement. These effects were also obtained in samples from CLL patients with clinical resistance to ibrutinib and mutations at C481. NC-1 treatment further decreased baseline CD69 surface levels, completely abrogated its upregulation following IgM activation, decreased CLL cells migration toward SDF-1 and overcame stromal anti-apoptotic protection. In conclusion, our results indicate that targeting BTK using the PROTAC strategy could be a potential novel therapeutic approach for CLL.

Targeting BTK Signaling in the Microenvironment of Solid Tumors as a Feasible Cancer Therapy Option.

Simple SummaryProtein tyrosine kinase BTK is essential for B cell maturation and proliferation. Dysregulation of BTK signaling in B cells leads to B cell lymphoma. In addition to B cells, BTK is also expressed in other types of immune cells including MDSC, dendritic cells, mast cells and macrophages, all of which comprise the tumor microenvironment in solid cancers. Although BTK inhibitors have been FDA-approved as the front-line treatment for B cell malignancy CLL/SLL, studies have been reluctant to report on BTKs role within the tumor microenvironment during solid cancer development limiting the possibility of using these BTK inhibitors as an adjuvant treatment option for solid cancers. Here, we review BTK signaling within the cells found in the tumor microenvironment as well as summarizing clinical trials using BTK inhibitors which target the tumor microenvironment in an attempt to combat solid tumors.The cell environment plays a pivotal role in determining cellular outcome, as well as cancer initiation, progression, and dissemination. Within this environment, in addition to the structural components, such as the extracellular matrix, there are various types of cells surrounding the tumor cells. Communication among these cells and the tumor cells via signaling pathways is important for tumor growth. Originally discovered in patients with immunodeficiency X-linked gammaglobulinemia, the Bruton’s tyrosine kinase (BTK) signaling pathway, known for its role in B cell maturation, is critical to cancer cell proliferation, metastasis and evasion of cancer eliminating cells. Given that BTK inhibitors have been FDA approved for chronic lymphocytic leukemia/small lymphocytic lymphoma and that the majority of BTK studies have been focused on B cells, the use of BTK inhibitors as a future treatment strategy of solid tumors has yet to be evaluated. In this review, we summarize studies analyzing BTK signaling within the cells found in the tumor microenvironment, as well as clinical trial where BTK inhibitors are currently being used to target the tumor microenvironment as a way to combat solid tumors.

Targeting Bruton Tyrosine Kinase With Zanubrutinib for Treatment of Vitreoretinal Lymphoma: Report of 3 Cases.

Vitreoretinal lymphoma (VRL) is a rare intraocular malignancy, and standard treatment approaches have not been defined yet. Bruton tyrosine kinase inhibitors are found to be effective in the treatment of primary central nervous system diffuse large B cell lymphoma. Herein, we retrospectively reported the efficacy and safety profiles of bruton tyrosine kinase inhibitors in three consecutive patients with VRL. All three cases of VRL occurred in patients with pre-treated primary central nervous system lymphoma and the central nervous system was not involved at the time of VRL diagnosis. They were treated with zanubrutinib, a bruton tyrosine kinase inhibitor, at 160 mg twice daily orally. Rapid improvement of visual acuity and tumor control was achieved in all involved eyes of these 3 patients. Complete remission was confirmed by fundus photograph and optical coherence tomography, and the level of interleukin-10, a well-recognized biomarker for vitreoretinal lymphoma, decreased to normal in all patients. Zanubrutinib was well tolerated in all three patients, and only one adverse event of grade 3 hypertension occurred, which resolved after adjusting antihypertensive drugs. As of March 2021, these three patients have been treated with zanubrutinib for 9 months, 7 months, and 6 months, respectively, and all remained in complete remission. In conclusion, targeting bruton tyrosine kinase with zanubrutinib in vitreoretinal lymphoma is feasible and our findings can be a foundation for a paradigm shift in treatment options for this rare disease. A prospective phase 2 study evaluating the efficacy and safety of zanubrutinib in patients with vitreoretinal lymphoma is ongoing to validate our findings (ChiCTR2000037921).

Discovery of potent and selective reversible Bruton’s tyrosine kinase inhibitors

Bruton’s tyrosine kinase (BTK) is a cytoplasmic, non-receptor tyrosine kinase member of the TEC family of tyrosine kinases. Pre-clinical and clinical data have shown that targeting BTK can be used for the treatment for B-cell disorders. Here we disclose the discovery of a novel imidazo[4,5-b]pyridine series of potent, selective reversible BTK inhibitors through a rational design approach. From a starting hit molecule 1, medicinal chemistry optimization led to the development of a lead compound 30, which exhibited 58 nM BTK inhibitory potency in human whole blood and high kinome selectivity. Additionally, the compound demonstrated favorable pharmacokinetics (PK), and showed potent dose-dependent efficacy in a rat CIA model.

Phase 2 study of ibrutinib in classic and variant hairy cell leukemia

Hairy cell leukemia (HCL) is a rare B-cell malignancy, and there is a need for novel treatments for patients who do not benefit from purine analogs. Ibrutinib, an oral agent targeting Bruton tyrosine kinase in the B-cell receptor signaling pathway, is highly effective in several malignancies. Its activity in HCL was unknown, so we conducted a multisite phase 2 study of oral ibrutinib in patients with either relapsed classic or variant hairy cell leukemia. The primary outcome measure was the overall response rate (ORR) at 32 weeks, and we also assessed response at 48 weeks and best response during treatment. Key secondary objectives were characterization of toxicity and determination of progression-free survival (PFS) and overall survival (OS). Thirty-seven patients were enrolled at 2 different doses (24 at 420 mg, 13 at 840 mg). The median duration of follow-up was 3.5 years (range, 0-5.9 years). The ORR at 32 weeks was 24%, which increased to 36% at 48 weeks. The best ORR was 54%. The estimated 36-month PFS was 73% and OS was 85%. The most frequent adverse events were diarrhea (59%), fatigue (54%), myalgia (54%), and nausea (51%). Hematologic adverse events were common: anemia (43%), thrombocytopenia (41%), and neutropenia (35%). Ibrutinib can be safely administered to patients with HCL with objective responses and results in prolonged disease control. Although the initial primary outcome objective of the study was not met, the observation of objective responses in heavily pretreated patients coupled with a favorable PFS suggests that ibrutinib may be beneficial in these patients. This trial was registered at www.clinicaltrials.gov as #NCT01841723.

A Moving Target: Inactivating BTK Mutations as Drivers of Follicular Lymphoma.

Drugs that target Bruton tyrosine kinase (BTK) have been highly successful and changed the landscape of therapies in B-cell lymphomas. However, their lower rates of effectiveness in follicular lymphoma are unexplained. Recent work describes inactivating BTK mutations that show that at least some follicular lymphomas do not require BTK.See related article by Hu et al., p. 2301.

Nx-2127, a Degrader of BTK and IMiD Neosubstrates, for the Treatment of B-Cell Malignancies

Bruton's tyrosine kinase (BTK) plays a key role in cell survival in B cell malignancies, such as chronic lymphocytic leukemia (CLL). Covalent inhibitors of BTK, such as ibrutinib and acalabrutinib, while effective, have been associated with the occurrence of resistance mutations. The most prevalent site of mutation, C481, renders covalent BTK inhibitors unable to form a covalent bond with BTK leading to diminished efficacy and disease progression. Small molecule-induced protein degradation offers a unique approach to target BTK for the treatment of B-cell malignancies. Chimeric Targeting Molecules (CTMs) catalyze ubiquitylation and proteasomal degradation of target proteins and are comprised of a ubiquitin ligase binding element ("harness"), a linker, and a target binding element ("hook"). NX-2127 is a CTM that contains a BTK hook linked to a cereblon (CRBN) harness. NX-2127 degrades 50% of cellular BTK (DC50) at &amp;lt; 5 nM across multiple cancer cell lines and in human PBMCs. BTK CTMs impair viability in the BTK-dependent ABC-DLBCL cell line, TMD8 (EC50: &amp;lt; 15 nM after 72 hours). Importantly, NX-2127 induces degradation of the mutated BTK-C481S in cells and inhibits proliferation of BTK-C481S mutant TMD8 cells more effectively than ibrutinib (NX-2127 EC50 values of &amp;lt; 30 nM versus &amp;gt; 1 μM for ibrutinib). Oral administration of NX-2127 in mice leads to dose-proportional exposure in plasma and BTK degradation to &amp;lt;10% of baseline levels in circulating and splenic B cells. In both WT TMD8 and C481S mutant xenograft models, daily oral administration of NX-2127 resulted in superior tumor growth inhibition (TGI) as compared to ibrutinib. NX-2127 also demonstrates potent degradation of BTK in cynomolgus monkeys with oral administration. Following 14 days of once daily, oral dosing in cynomolgus monkey, BTK levels are suppressed to &amp;lt;10% of baseline levels at doses as low as 1 mg/kg. In addition to potent BTK degradation, NX-2127 possesses IMiD-like properties through the design of the CRBN binding harness that catalyzes the degradation of CRBN neosubstrates Aiolos (IKZF3) and Ikaros (IKZF1). This activity is associated with increased T cell activation and anti-tumor effects of the IMiD drugs lenalidomide and pomalidomide. In primary human T cells, NX-2127 catalyzes the degradation of Aiolos and Ikaros with of 25 nM and 54 nM, respectively, potencies which are similar to those of lenalidomide (20 nM and 343 nM, respectively). Corresponding with such degradation, NX-2127 stimulates T cell activation as measured by increased IL-2 production in primary human T Cells in a manner similar to lenalidomide and pomalidomide. The dual activity of BTK degradation combined with immunomodulation of NX-2127 supports its development for the treatment of B-cell malignancies. Disclosures Robbins: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Kelly:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tan:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. McIntosh:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Wu:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Konst:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Kato:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Peng:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mihalic:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Weiss:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Perez:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tung:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Kolobova:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Borodovsky:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rountree:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tenn-McClellan:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Noviski:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ye:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Basham:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ingallinera:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. McKinnell:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Karr:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Powers:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Guiducci:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sands:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company.

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

AbstractDiffuse 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-β expression was demonstrated to drive resistance in ibrutinib-resistant cells, and resistance was reversed by the blocking activity of PI3K-β/δ. Treatment with the selective PI3K-β/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.