Ibrutinib-resistant Chronic Lymphocytic Leukemia Research Articles

Single-Cell RNA-Seq Analysis Reveals Distinct Tumor and Immunosuppressive T Cell Phenotypes in CLL Patients Treated with Ibrutinib

Introduction: The development of Bruton tyrosine kinase inhibitors (BTKIs) and their introduction into clinical practice represents a major advance in the treatment of chronic lymphocytic leukemia (CLL). Monotherapy with ibrutinib or other BTKIs generally do not induce complete remissions or undetectable minimal residual disease (uMRD) even with extended therapy. The reason why some patients relapse, and some have minimal residual disease is unknown. Therefore, there is a need to understand the differences between ibrutinib sensitive and resistant CLL cells along with the immune microenvironment to identify novel therapeutic targets. Methods: Here, we investigate the cellular heterogeneity of peripheral blood mononuclear cells from patients with CLL treated with ibrutinib using single-cell RNA sequencing. To understand ibrutinib resistance mechanisms, samples were collected from 8 patients at baseline (before treatment) (1), where BTK C481S is present with low variant allele frequency (VAF) (2) and at time of relapse (3). Data from these were compared to ibrutinib sensitive patient samples at baseline (1) (before treatment), 3yr (2) and 5yr (3) on treatment timepoints. Single cells were isolated using the 10X Genomics 5' immune profiling kit. Data were processed with CellRanger v3.1.0. Poor quality cells were filtered out based on a high proportion of mitochondrial reads and low overall feature count. Sample variation was reduced using Batchelor to aid clustering. Clustering was performed using the partitioning and Leiden methods. Cluster top markers were calculated using functions from monocle3. Identity mapping was performed using Seurat and a published PBMC reference dataset. Results: Single-cell RNA sequencing revealed transcriptional heterogeneity within the B cell cluster (Fig 1A). Three subpopulations of B cells were defined based on differential representation in the ibrutinib-sensitive and resistant patients. Ibrutinib sensitive cells showed enrichment of B cell populations with upregulation of MHC I molecules and TNF family members. We also identified that inflammatory response and metabolic-related pathways were decreased, whereas cellular response to stress and DNA repair programs were increased in the ibrutinib resistance samples. Gene module analysis showed that ibrutinib sensitive samples had an increase in T cell activation, RNA splicing, cell adhesion and migration programs during therapy. We hypothesized that the emergence of BTK C481S mutation in ibrutinib-resistant cells would lead to a distinct transcriptional profile. Long-read sequencing was performed using Oxford Nanopore technology. BTK-mutant cells were identified based on the presence of two or more unique RNA molecules detected with the C481S mutation and these were predominantly identified in the low-VAF and relapse samples. However, there was no significant difference between BTK-mutant cells and the remaining B cells. To see if T cell phenotypes reflected the differences observed in B cell transcriptional state, we characterized and quantified T cell populations according to ibrutinib response and investigated the changes in the T cell population. Ibrutinib markedly increased CD4+ and CD8+ T cell numbers in both resistant and sensitive CLL patients. However, there was a significant (p<0.05) enrichment of Tregs in the ibrutinib-resistant samples. We found a connection between the immunosuppressive phenotype in the microenvironment demonstrating tumor's intrinsic immunological reactive program with exhausted T cells. Also, we found significant difference in the expression of checkpoint molecules and suppressive genes in T cells (Fig 1B) and NK cells. Conclusion: Our findings demonstrate that the B cell population in ibrutinib-resistant and ibrutinib sensitive CLL patients exhibit distinct transcriptional states. At the single-cell level, our findings demonstrate a systematic picture of diverse malignancy and microenvironmental changes in CLL. Overall, these findings provide an insight into the complex molecular and cellular interactions of CLL that may have a crucial role in BTKI resistance.

Curcumin Enhances Ibrutinib Induced Killing Effect Against TP53-Mutated Chronic Lymphocytic Leukemia Cells in Vitro

Introduction TP53 mutation and del (17p) are the most important poor prognostic factors in patients with Chronic Lymphocytic Leukemia (CLL).Patients with deletion 17p or TP53 mutations tend to have lower response rates to standard first-line chemoimmunotherapy and a more aggressive clinical course(Stilgenbauer et. al. Blood 2014). RESONATE-2 study long-term follow-up showing that ibrutinib continues to demonstrate significant and durable clinical benefit in older patients, including those with TP53 mutation(Barr et. al. Blood Adv 2022).However, CLL bearing del(17p) showed inferior OS and time to next treatment compared to non-del(17p)(Mato et. al. Haematologica 2022). A significant proportion of patients treated with single-agent ibrutinib experienced CLL progression.BTK and/or PLCG2 mutations were present in 80.0% of patients progressing with CLL, confirming prior reports on the high prevalence of these mutations in patients with ibrutinib-resistant CLL(Ahn et. al. Blood 2017). Curcumin is a bioactive natural polyphenol compound extracted from the Curcuma longa(Zingiberaceae plant). Curcumin has anti-tumor effect and can induce apoptosis and inhibit the proliferation of a series of tumor cells(Willenbacher et. al. Int J Mol Sci 2019). Curcumin may limit the development of therapy-related toxicity and reduce the financial burden of treatment.In this study, we found that Curcumin inhibits TP53-mutated CLL cells, and eliciting a strong synergistic cytotoxic effect in combination with ibrutinib. Methods The inhibitory effect of ibrutinib combined with curcumin on the proliferation of TP53-mutated Chronic Lymphocytic Leukemia cells was detected by CCK8, and the apoptosis of TP53-mutated CLL cells induced by ibrutinib combined with curcumin was detected by flow cytometry;The differences of gene and signal pathway expression among different treatment groups were analyzed by RNA-seq ;Using Western blot technology to verify the expression of related pathway proteins and explore the mechanism of curcumin increasing the killing effect of ibrutinib against TP53-mutated Chronic Lymphocytic Leukemia cells. Results The CCK8 results showed that the IC50 of ibrutinib for 24 hours was 40.61±2.35μM, while the IC50 of curcumin for 24 hours was 26.6±0.94μM. The IC50 of ibrutinib for 48 h was 14.6 ±0.8307μM, and the IC50 of curcumin for 48 h was 15.7±2.53μM.Cell inhibition of various group for 48h is shown in Figure 1.The CI of ibrutinib combined with curcumin at 24 hours and 48 hours was calculated by CompuSyn software. The results showed that the CI values of ibrutinib combined with curcumin were less than 1 at 24 h and 48 h, suggesting that ibrutinib combined with curcumin has a synergistic killing effect on CLL cells with TP53 mutation;Flow cytometry showed that the IC50 of ibrutinib for 24 hours was 67.20±2.86μM, while the IC50 of curcumin for 24 hours was 144±6.6 μM. The CI value of ibrutinib combined with curcumin was less than 1 at 24 h, suggesting that ibrutinib combined with curcumin has a synergistic promoting apoptosis on CLL cells with TP53 mutation;RNA-seq analysis and KEGG analysis of down-regulated genes of combination group compared with the control group were mainly enrich in PI3K/AKT, Cytokine-Cytokine Recptor signal pathway and so on;4.The results of Western blot showed that the protein expression levels of P65PI3K and p53 in the single drug group and the combination group were down-regulated in varying degrees, especially in the combination group（Figure 2). Conclusion Ibrutinib combined with curcumin has synergistic killing effect on TP53- mutated CLL cells;Curcumin increases the killing effect of ibrutinib on TP53-mutated CLL cells by inhibiting PI3K and NFκB pathway, and degrading p53mt protein.

Pirtobrutinib targets BTK C481S in ibrutinib-resistant CLL but second-site BTK mutations lead to resistance.

Covalent inhibitors of Bruton tyrosine kinase (BTK) have transformed the therapy of chronic lymphocytic leukemia (CLL), but continuous therapy has been complicated by the development of resistance. The most common resistance mechanism in patients whose disease progresses on covalent BTK inhibitors (BTKis) is a mutation in the BTK 481 cysteine residue to which the inhibitors bind covalently. Pirtobrutinib is a highly selective, noncovalent BTKi with substantial clinical activity in patients whose disease has progressed on covalent BTKi, regardless of BTK mutation status. Using invitro ibrutinib-resistant models and cells from patients with CLL, we show that pirtobrutinib potently inhibits BTK-mediated functions including B-cell receptor (BCR) signaling, cell viability, and CCL3/CCL4 chemokine production in both BTK wild-type and C481S mutant CLL cells. We demonstrate that primary CLL cells from responding patients on the pirtobrutinib trial show reduced BCR signaling, cell survival, and CCL3/CCL4 chemokine secretion. At time of progression, these primary CLL cells show increasing resistance to pirtobrutinib in signaling inhibition, cell viability, and cytokine production. We employed longitudinal whole-exome sequencing on 2 patients whose disease progressed on pirtobrutinib and identified selection of alternative-site BTK mutations, providing clinical evidence that secondary BTK mutations lead to resistance to noncovalent BTKis.

Duvelisib Eliminates CLL B Cells, Impairs CLL-Supporting Cells, and Overcomes Ibrutinib Resistance in a Xenograft Model.

Inhibitors of Bruton's tyrosine kinase (BTKi) and PI3K (PI3Ki) have significantly improved therapy of chronic lymphocytic leukemia (CLL). However, the emergence of resistance to BTKi has introduced an unmet therapeutic need. Hence, we sought evidence for essential roles of PI3K-δi and PI3K-γi in treatment-naïve and BTKi-refractory CLL. Responses to PI3K-δi, PI3K-γi, and the dual-inhibitor duvelisib in each B, T, and myeloid cell compartments of CLL were studied in vitro, and in a xenograft mouse model using primary cells from treatment-naïve and ibrutinib-resistant patients, and finally, in a patient with ibrutinib-resistant CLL treated with duvelisib. We demonstrate the essential roles of PI3K-δ for CLL B-cell survival and migration, of PI3K-γ for T-cell migration and macrophage polarization, and of dual inhibition of PI3K-δ,γ for efficacious reduction of leukemia burden. We also show that samples from patients whose disease progressed on ibrutinib were responsive to duvelisib therapy in a xenograft model, irrespective of BTK mutations. In support of this, we report a patient with ibrutinib-resistant CLL, bearing a clone with BTK and PLCγ2 mutations, who responded immediately to single-agent duvelisib with redistribution lymphocytosis followed by a partial clinical remission associated with modulation of T and myeloid cells. Our data define the mechanism of action whereby dual inhibition of PI3K-δ,γ affects CLL B-cell numbers and T and myeloid cell pro-leukemia functions and support the use of duvelisib as a valuable approach for therapeutic interventions, including for patients refractory to BTKi.

Abstract 4933: Duvelisib eliminates CLL B Cells, impairs CLL-supporting cells, and overcomes ibrutinib resistance in a patient-derived xenograft model

Abstract Duvelisib (IPI-145), a first-in-class, oral, dual PI3K-δ,γ inhibitor, has been approved by the FDA for the treatment of patients with chronic lymphocytic leukemia (CLL), small lymphocytic and follicular lymphoma. Duvelisib potently inhibits PI3K-δ and PI3K-γ, thereby offering a novel therapeutic approach. Here we aimed to understand how duvelisib targets CLL cells and the tumor microenvironment (TME) and hence how it affects CLL, even with BTK-resistant disease. We first assessed the distinct contributions of PI3K-δ and PI3K-γ on CLL cell survival, proliferation, and migration in vitro by using PI3K-δ (PI3K-δi) or PI3K-γ (PI3K-γi) specific inhibitors in addition to duvelisib. We found a significant reduction in viable CLL cells after exposure to PI3K-δi and duvelisib. By measuring Ki67 and p-AKT levels, we found duvelisib and PI3K-δi more potently block CLL cell proliferation than PI3K-γi. PI3K-δi and duvelisib also significantly reduced the migration of CLL B cells into the spleen relative to control. In contrast, PI3K-γi inhibited T cell not B cell homing in vitro and in vivo. We then investigated the impact of single versus dual inhibitory agents on myeloid cells. Tumor-associated macrophages (TAMs) of the “M2 phenotype” contribute to a pro-tumor TME by preventing the induction of T cell mediated anti-tumor immunity. We expanded murine bone marrow derived monocytes and then polarized them to M2 macrophages, in the absence or presence of PI3Ki. At 100nM, PI3K-γi, but not PI3K-δi, significantly inhibited M2 polarization. Duvelisib significantly reduced Arg1 expression at doses equal to or above 10nM. While co-culturing leukemic B cells with M2-polarized murine macrophages increased CLL-cell survival, addition of duvelisib to such co-cultures significantly reduced CLL cell survival. Altogether, duvelisib sensitizes primary CLL cells to apoptosis and abrogates M2 cell-mediated CLL-cell survival. Finally, in a patient-derived xenograft mouse model (PDX), we demonstrated the essential roles of PI3K-δ and PI3K-γ in the CLL TME. Specifically, we found a more efficacious inhibition in CLL cell burden by dual inhibition of PI3K-δ,γ. Also, samples from patients whose disease progressed on ibrutinib were responsive to duvelisib therapy in PDX, irrespective of BTK mutations. In support of this, we identified an ibrutinib resistant CLL patient, with a clone exhibiting BTK and PLCγ2 mutations who responded immediately to single agent duvelisib with redistribution lymphocytosis followed by a partial clinical remission associated with subsequent modulation of T and myeloid cells. In conclusion, our data define the mechanism of action whereby dual inhibition of PI3K-δ,γ affects CLL B cell numbers and T and myeloid cell pro-leukemia functions, supporting the use of duvelisib as a potentially valuable therapeutic intervention, including for patients refractory to BTKi. Citation Format: Shih-Shih Chen, Jacqueline C. Barrientos, Gerardo Ferrer, Priyadarshini Ravichandran, Michael Ibrahim, Yasmine Kieso, Jeffery L. Kutok, Marisa Peluso, Sujata Sharma, David T. Weaver, Jonathan A. Pachter, Kanti R. Rai, Nicholas Chiorazzi. Duvelisib eliminates CLL B Cells, impairs CLL-supporting cells, and overcomes ibrutinib resistance in a patient-derived xenograft model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4933.

Longitudinal Omics Data Followed By Preclinical Treatment Studies Identify Proteasome Inhibition As Effective Therapy for Ibrutinib-Resistant Chronic Lymphocytic Leukemia

Resistance to ibrutinib treatment is an increasing problem for patients with chronic lymphocytic leukemia (CLL) as it leads to dismal clinical outcomes. Acquired mutations in the therapy target BTK or its downstream mediator PLCG2 explain only partly the non-responsiveness to this treatment, as merely a subset of patients harbor such mutations, or if so, only in a fraction of the malignant B cells. Besides genetic aberrations, molecular changes both CLL cell-intrinsic and -extrinsic have been associated with ibrutinib resistance. But so far these alterations have not led to the development of novel therapy approaches for patients who are non-responsive to ibrutinib treatment. We have generated a mouse model of ibrutinib resistance by treating mice upon adoptive transfer of Eµ-TCL1 leukemia (TCL1 CLL) continuously with ibrutinib. For a period of several weeks, the leukemic mice respond very well to the treatment, but similarly as observed in patients, relapse under therapy occurs with an aggressive outgrowth of the malignant cells (Figure A), which are non-responsive to ibrutinib upon retransplantation. By whole exome sequencing (WES) of TCL1 CLL cells before treatment and after relapse we did not detect any mutations in Btk or downstream signaling molecules in the resistant cells. In addition, the identified mutations were of low allele frequency and no recurrent mutations were detectable in the relapse samples that could explain the non-responsiveness to ibrutinib. To analyze molecular adaptations that drive resistance to ibrutinib, we performed transcriptome and proteome analyses of TCL1 CLL cells from mice treated with ibrutinib or vehicle at an early responsive time point, and a late time point, when mice got refractory to treatment with ibrutinib (see arrows in Figure A). Comparative analyses of these complex data sets suggested alterations in the proteasome activity as a driver of ibrutinib resistance. By quantifying total ubiquitination of proteins, we further confirmed reduced levels of K48-ubiquitination which suggest an increased proteasomal activity in ibrutinib-resistant tumors. Based on these results, we performed preclinical treatment studies with the proteasome inhibitor carfilzomib, which more efficiently controlled TCL1 CLL in ibrutinib-resistant than in ibrutinib-sensitive tumors. Most interestingly, when carfilzomib was added as salvage therapy at the time of ibrutinib-resistance development in mice, we observed a clear survival benefit compared to mice who continued on ibrutinib monotherapy (Figure B). To verify the relevance of these findings for patients with ibrutinib-resistant CLL, we acquired proteome data of CLL cells from three sequential time points in 3 patients: (1) before start of ibrutinib treatment, (2) during ibrutinib responsiveness (within the first 6 months of treatment), (3) upon ibrutinib refractoriness. This allowed us to identify common dynamic clusters of deregulated proteins that we are currently exploring concerning their role in resistance development. In addition, we treated CLL cells from ibrutinib-resistant patients cultured under lymph node-mimicking conditions ex vivo with several proteasome inhibitors and showed effective responses of these cells comparable to CLL samples of ibrutinib-sensitive patients. Altogether, our omics data approach identified proteasome inhibitors as attractive novel treatment options for CLL patients that do not respond to ibrutinib. Current investigations of proteome data acquired from TCL1 CLL samples before and during carfilzomib treatment aim at identifying the molecular targets of the proteasome with relevance for ibrutinib resistance. Figure:A) Treatment response in mice with TCL1 CLL to ibrutinib (Ibr) and vehicle (Veh) measured as the number of CD5+ CD19+ CLL cells in blood over time. B) Survival curves of mice with TCL1 CLL treated either with vehicle or ibrutinib as monotherapy, or a combination starting with 2 weeks of ibrutinib treatment, followed by the addition of carfilzomib (CFZ). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Novel Triple-Action Inhibitor Targeting B-Cell Receptor Signaling and BRD4 Demonstrates Preclinical Activity in Chronic Lymphocytic Leukemia.

B-cell chronic lymphocytic leukemia (CLL) results from intrinsic genetic defects and complex microenvironment stimuli that fuel CLL cell growth through an array of survival signaling pathways. Novel small-molecule agents targeting the B-cell receptor pathway and anti-apoptotic proteins alone or in combination have revolutionized the management of CLL, yet combination therapy carries significant toxicity and CLL remains incurable due to residual disease and relapse. Single-molecule inhibitors that can target multiple disease-driving factors are thus an attractive approach to combat both drug resistance and combination-therapy-related toxicities. We demonstrate that SRX3305, a novel small-molecule BTK/PI3K/BRD4 inhibitor that targets three distinctive facets of CLL biology, attenuates CLL cell proliferation and promotes apoptosis in a dose-dependent fashion. SRX3305 also inhibits the activation-induced proliferation of primary CLL cells in vitro and effectively blocks microenvironment-mediated survival signals, including stromal cell contact. Furthermore, SRX3305 blocks CLL cell migration toward CXCL-12 and CXCL-13, which are major chemokines involved in CLL cell homing and retention in microenvironment niches. Importantly, SRX3305 maintains its anti-tumor effects in ibrutinib-resistant CLL cells. Collectively, this study establishes the preclinical efficacy of SRX3305 in CLL, providing significant rationale for its development as a therapeutic agent for CLL and related disorders.

Abstract 5432: Targeted alpha therapy with 212Pb-NNV003 in treatment of NHL

Abstract Introduction: CD37 has shown promise as a new therapeutic target for B-cell malignancies. CD37 is highly and selectively expressed on the surface of mature B lymphocytes and B-cell malignancies. Alpha-emitting radionuclides have demonstrated potential for cancer targeted therapies because of efficient energy deposition along the short alpha track (50-100 µm) causing localized and irreparable DNA damage while sparing surrounding healthy tissues. We have developed a targeted alpha therapy (TAT) where the CD37-specific antibody NNV003 is coupled to the alpha-particle-generating radioisotope 212Pb for the treatment of non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Approximately 90,000 cases of CLL and NHL are expected annually in the US. Standard of care includes chemotherapy combined with anti-CD20 antibodies (obinutuzumab and rituximab) and Bruton’s tyrosine kinase inhibitor ibrutinib. While these therapies are initially effective, most patients inevitably relapse. Materials and methods: The efficacy and tolerability of a single dose 212Pb-NNV003 treatment were evaluated in disseminated models using CD37-expressing human CLL (MEC-2) and Burkitt’s lymphoma (Daudi) cells. Comparison to therapeutic doses of ibrutinib was investigated in the MEC-2 model. R2G2 or CB17-SCID mice were injected intravenously with 2.5×106 MEC-2 or 10×106 Daudi cells on day 0, followed by intravenous injection of 212Pb-NNV003 (2.5, 5 or 7.5 µCi in Daudi model and 5, 10, 15 or 20 µCi in MEC-2 model) or daily oral administration of ibrutinib (12.5 or 25 mg/kg) starting on day 2 (n=10-12 per group). 212Pb-cetuximab (unspecific isotype control), unlabelled NNV003 and NaCl were used as controls. Results: In vitro testing of the antibody showed that MEC-2 cells were 5 times more resistant to ibrutinib than Daudi cells. Treatment of mice injected intravenously with Daudi cells with 2.5 μCi 212Pb-NNV003 resulted in survival of 100% of the mice 100 days after start of treatment, while 100 days after start of treatment with 100 μg obinutuzumab only 40% of the mice were still alive. Control mice had a median survival of 7-8 weeks. Treatment of mice injected intravenously with MEC-2 cells with 15 μCi 212Pb-NNV003 resulted in survival of 90-100% for more than 25 weeks in two independent experiments. Ibrutinib treated mice with intravenous MEC-2 cells presented a median survival of only 4.3-4.4 weeks, comparable to the unlabelled NNV003 and saline groups (4.1 and 4.4 weeks). Mice receiving non-specific 212Pb-cetuximab presented a median survival of 7-9 weeks. Conclusion: The study shows that a single injection 212Pb-NNV003 is safe and effective for the treatment of CD37 positive CLL and NHL in preclinical models, with promising efficacy in both ibrutinib-resistant and sensitive CLL models. Treatment with 212Pb-NNV003 was superior to both ibrutinib and obinutuzumab. Further clinical testing is warranted. Citation Format: Jostein Dahle, Amal Saidi, Tania Stallons, Helen Heyerdahl, Ada Repetto-Llamazares, Julien Torgue. Targeted alpha therapy with 212Pb-NNV003 in treatment of NHL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5432.

Targeting PAK1 Overcomes Resistance to Ibrutinib in Chronic Lymphocytic Leukemia

Objective:Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder that mainly affects the elderly and is characterized by the expansion of small mature B-cells. New targeted drugs, such as the BTK inhibitor ibrutinib, have greatly improved patient survival but have also posed the challenge of drug resistance. The three-dimensional (3D) spatial structure of chromatin is highly dynamic and varies greatly between cell types and developmental stages, with the maintenance of chromatin homeostasis being of major significance in disease prevention. Accumulating evidence has suggested that changes in 3D genomic structures play an important role in cell development and differentiation, disease progression, as well as drug resistance. Nevertheless, the characteristics and functional significance of chromatin conformation in the resistance of CLL to ibrutinib remain unclear. In this study, we aimed to investigate the mechanism underlying ibrutinib resistance through multi-omics profiling, including the study of chromatin conformation. Thus, we would be able to demonstrate the importance of chromatin spatial organization in CLL and highlight the oncogenic factors contributing to CLL development and mediating ibrutinib resistance.Methods:An ibrutinib-resistant cell line was established by exposing cells to increasing doses of ibrutinib. High-throughput chromosome conformation capture (Hi-C), assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), bulk RNA sequencing (RNA-seq), and Tandem Mass Tag (TMT) were performed to explore differences between ibrutinib-resistant and parental cells. Peripheral blood mononuclear cells (PBMCs) from 53 CLL patients were collected for RNA-seq. Mitochondrial respiration and glycolysis were assessed via Seahorse analysis. The growth-inhibitory effects of tested drugs were evaluated via a CCK8 assay, and the combination index (CI), indicating synergy, was calculated using CompuSyn software. Apoptosis was detected via annexin V staining.Results:Between ibrutinib-resistant and parental cells changes in some chromosomes, including chr11 were observed (Figure 1A). p21-activated kinase 1 (PAK1), which is located on chr11 and frequently overexpressed or excessively activated in almost all cancer types and involved in almost every stage of cancer progression, was first explored for its role in CLL progression and drug resistance. The oncogene PAK1 was observed locate in a region where B-to-A compartment switching occurred (Figure 1B). Consistent with the results of ATAC-seq, RNA-seq, and TMT, Hi-C analysis revealed a transcriptional upregulation of PAK1 in ibrutinib-resistant CLL cells (Figure 1C). Functional analysis demonstrated that PAK1 overexpression significantly promoted cell proliferation, while knockdown markedly suppressed cell viability (Figure 1D). Cell viability assays indicated that the depletion of PAK1 increased ibrutinib sensitivity (Figure 1E). In addition, PAK1 positively regulates glycolysis and oxidative phosphorylation in CLL cells (Figure 1F and G).To verify the results of sequencing and further explore the role of PAK1 in CLL, B-cells from healthy volunteers and PBMCs from CLL patients were collected. The level of PAK1 mRNA expression was significantly higher in CLL primary cells than in B-cells from healthy volunteers (Figure 1H). Kaplan-Meier survival analysis of qRT-PCR data confirmed that patients with high PAK1 expression had a significantly lower OS (Figure 1I).IPA-3, the small molecular inhibitor of PAK1 suppressed the proliferation of ibrutinib-resistant and parental CLL cells in a dose-dependent manner. The combination of IPA-3 and ibrutinib exerted potent cell growth inhibition (Figure 1J), and the combination index (CI) calculated using the CompuSyn software confirmed the synergistic effect (CI<1) of this combinatorial therapy (Figure 1K).Conclusions:In the current study, we have provided a genome-wide view of alterations in 3D chromatin organization between ibrutinib-resistant and parental CLL cells and confirmed the oncogenic role of PAK1 in CLL. Most importantly, our research provides promising therapeutic targets for overcoming ibrutinib resistance. In particular, the treatment of CLL patients with a combination of IPA-3 and ibrutinib may improve clinical outcomes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Single-Cell RNA Sequencing Suggests Novel Drivers of Chronic Lymphocytic Leukemia Patients with Ibrutinib Resistance

Objective:Ibrutinib is currently the most widely used BTK inhibitor that approved for the treatment of both initially diagnosed and relapsed and refractory chronic lymphocytic leukemia (CLL) patients. Although ibrutinib shows high response rates in clinical practice, it has certain limitations. There are still a certain number of patients who have to discontinue treatment due to drug-resistance or side effects. The ibrutinib resistance of CLL patients has caused widespread concerns, necessitating the development of novel treatment strategies.Methods:Here, we examined the heterogeneity of peripheral blood mononuclear cells (PBMCs) from patients with ibrutinib-sensitive (IBS) and -resistant (IBR) CLL by analyzing bulk and single-cell level gene expression profiles, clinical features, biological properties, and phenotypes. Seven distinct ibrutinib-resistant subpopulations were identified and two candidate genes LGALS1 (galectin 1, Gal-1) and LAG3 (lymphocyte-activating gene 3, CD223) were screened that contribute toward ibrutinib-resistance and poor survival in CLL patients. These results were validated in primary cells from CLL patients and also in ibrutinib-resistant CLL cell line (MEC1-IR) which was generated by culturing the parental cell line in vitro with progressively increasing concentrations of ibrutinib. Marker-gene expression was detected using qRT-PCR, western blotting, and ELISA, while functional analyses including CCK8, flow cytometry and trypan blue staining were conducted with or without OTX008, a selective Gal-1 inhibitor.Results:ScRNA-seq revealed that cells from IBR and IBS samples were distributed in different clusters and suggested that IBR cells display a unique transcriptional pattern (Fig A). IBR-B cells have higher stemness scores and are enriched in some energy metabolismPathways (Fig B). According to the proportion of B cells from IBR samples, we classified each B-cell cluster into three main subgroups, i.e., IBR, IBS, and shared cluster (Fig C). IBR-B cells displayed more interactions with monocytes, NK, T, and dendritic cells than IBS B cells, suggesting that IBR B cells may actively build connections with other immune cells to reshape the protective niche (Fig D). A close correlation between LGALS1 and LAG3 expression was observed and both of them were found to be highly expressed in IBR CLL patients (Fig E), their expression level gradually increased along the trajectory of B cells from IBS to IBR (Fig F). Diagnosis and prognosis stratification of CLL with receiver operating characteristic (ROC) curves revealed that patients with higher expression of both LGALS1 and LAG3 showed the poorest overall survival, indicating that LGALS1 and LAG3 are associated with ibrutinib-resistance and poor prognosis in CLL (Fig G). Concordantly, acquired resistance following chronic exposure to ibrutinib leads to upregulation of LGALS1 and LAG3 (Fig H). LGALS1 inhibitor OTX008 effectively inhibits the growth of ibrutinib-resistant CLL cells, particularly for IBR patients (Fig I).Conclusion:In conclusion, our findings demonstrate that ibrutinib-resistant CLL cells exhibit a unique transcriptional pattern. The combination of LGALS1 and LAG3 expression could serve as an indicator of the sensitivity of ibrutinib and prognosis of CLL patients. LGALS1 inhibitor OTX008 helps to overcome ibrutinib-resistance of CLL cells. Our findings may expand the current knowledge regarding ibrutinib-resistant CLL patients, identify improved biomarkers for patient selection, and offer a promising combinatorial therapeutic strategy for IBR CLL patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Development and characterization of prototypes for in vitro and in vivo mouse models of ibrutinib-resistant CLL

AbstractAlthough ibrutinib improves the overall survival of patients with chronic lymphocytic leukemia (CLL), some patients still develop resistance, most commonly through point mutations affecting cysteine residue 481 (C481) in Bruton's tyrosine kinase (BTKC481S and BTKC481R). To enhance our understanding of the biological impact of these mutations, we established cell lines that overexpress wild-type or mutant BTK in in vitro and in vivo models that mimic ibrutinib-sensitive and -resistant CLL. MEC-1 cell lines stably overexpressing wild-type or mutant BTK were generated. All cell lines coexpressed GFP, were CD19+ and CD23+, and overexpressed BTK. Overexpression of wild-type or mutant BTK resulted in increased signaling, as evidenced by the induction of p-BTK, p-PLCγ2, and p-extracellular signal–related kinase (ERK) levels, the latter further augmented upon IgM stimulation. In all cell lines, cell cycle profiles and levels of BTK expression were similar, but the RNA sequencing and reverse-phase protein array results revealed that the molecular transcript and protein profiles were distinct. To mimic aggressive CLL, we created xenograft mouse models by transplanting the generated cell lines into Rag2−/−γc−/− mice. Spleens, livers, bone marrow, and peripheral blood were collected. All mice developed CLL-like disease with systemic involvement (engraftment efficiency, 100%). We observed splenomegaly, accumulation of leukemic cells in the spleen and liver, and macroscopically evident necrosis. CD19+ cells accumulated in the spleen, bone marrow, and peripheral blood. The overall survival duration was slightly lower in mice expressing mutant BTK. Our cell lines and murine models mimicking ibrutinib-resistant CLL will serve as powerful tools to test reversible BTK inhibitors and novel, non–BTK-targeted therapeutics.

Predicting Mechanisms of Ibrutinib-Resistance in Chronic Lymphocytic Leukemia (CLL) Using Computational-Based Modeling for Personalized Therapy with Clinical Validation

Background: Bruton's tyrosine kinase (BTK), a mediator of B cell receptor signaling, has been effectively exploited as a therapeutic target in Chronic lymphocytic leukemia (CLL). Ibrutinib is a BTK inhibitor that has shown excellent efficacy in treatment-naïve, relapsed/refractory, and high-risk CLL. However, concerns regarding ibrutinib resistance have emerged and characterizing the underlying mechanisms may aid in identifying patients with increased risk of resistance/disease progression. Moreover, understanding the interplay between determinants of ibrutinib resistance may reveal alternate therapeutic targets. Using a computational biology modeling (CBM) simulation-based method, we reverse-engineered the salient molecular architecture of a CLL patient progressing on ibrutinib to identify potential drug combinations to overcome drug resistance and induce remission.Methods: For validation of the computational biological modeling (CBM) software (Cellworks group), we selected a cohort of 15 ibrutinib resistant CLL patients from previously published studies (Table 1). Genomic data derived from whole exome sequencing (WES), targeted next-generation sequencing (NGS), copy number variation (CNV), and/or karyotype data were used in this study. All available genomic data for each profile was entered into the CBM, which generates a disease-specific protein network map using PubMed and other online resources. Disease-biomarkers that are unique to each patient were identified within the protein network maps by using CBM technology. Ibrutinib was selected from a digital drug library and simulated on every relapsed CLL patient case. Drug impact was assessed by quantitatively measuring the drug's effect on a cell growth score, which is a composite of cell proliferation, viability, and apoptosis, along with the simulated impact on each disease-specific biomarker score. Each patient-specific map was digitally screened to identify any Ibrutinib resistance mechanism(s).Results: In our modeled patient set, 4 different resistance mechanisms were identified across 14/15 ibrutinib- refractory CLL cases. 1) BTK C481S mutation (n=4) impacts ibrutinib binding affinity; 2) PLCG2 mutations (n=4) lead to activation of PRKCB and AKT1 signaling; 3) CARD11 mutation (n=2) results in constitutive activation of BTK downstream effectors; and 4) decreased G-protein coupled receptor (GPCR) pathway signaling, an upstream activator of BTK (n=5), was found to be a novel resistance mechanism identified by CBM. Knockdown (KD) of ALOX5AP, ARRB1 and CYSLTR2 reduced GPCR pathway signaling, resulting in reduced BTK activity (Table 1). After validating the CBM algorithm, we rendered a digital avatar for a relapsed-refractory CLL patient (Del17p+); progressing on ibrutinib. The patient had rapidly escalating ALC and lymphadenopathy; with the largest node measuring ~16cm. In addition to p53 loss, simulation modeling accounted for BTK C481S mutation (BAF 0.69) expressed in the patients tumor cells. CBM predicted venetoclax would be the most effective therapeutic for the patient due to several upstream signaling pathways converging on Bcl-2. Indeed, addition of venetoclax to the ongoing ibrutinib monotherapy dramatically reduced the patients ALC (from 113 to 3.3 x 109/L) by day 4 post-treatment, accompanied by reduction in lymphadenopathy (left axillary node decreased to ~3cm). Ibrutinib was discontinued within 1 month of starting the combination, with venetoclax monotherapy continued per dosing guidelines thereafter. Notably, the patient maintained remission status on venetoclax monotherapy alone.Conclusions: Computational modeling was used to identify ibrutinib resistance mechanisms in CLL patients. Using this novel approach, we identified an effective therapy (venetoclax) for a high-risk CLL patient who was progressing on ibrutinib. CBM-based approaches can be used to predict alternative therapeutic regimens for drug-refractory patients, targeting the disease-specific biomarkers unique to each patient. Altogether, our study demonstrates proof-of-principle on the use of computational predictive algorithms to simulate multiple protein-interaction networks operational in a tumor cell to exploit individualized therapeutic vulnerabilities. [Display omitted] DisclosuresYsebaert:Janssen: Consultancy, Research Funding, Speakers Bureau. Kumar:Cellworks: Employment. Singh:Cellworks Research India Pvt. Ltd: Employment. Ullal:Cellworks Research India: Employment. Bhargav:Cellworks: Employment. Azam:cellworks: Employment. Abbasi:Cellworks Group Inc.: Employment. Vali:Cellworks Group Inc.: Employment. Cogle:Celgene: Other: Membership on Steering Committee for Connect MDS/AML Registry.

Discovery of HBW-3-10: A potent, orally active, reversible Bruton’s tyrosine kinase (BTK) inhibitor with antitumor activity in mice.

e15062 Background: The first generation irreversible BTK inhibitor ibrutinib has been approved for the treatment of B cell-related diseases, including chronic lymphocytic leukemia (CLL), for several years. However, CLL patients who used ibrutinib may develop drug resistance due to acquired mutations, in particular BTK C481S that directly impacts the binding of ibrutinib. In recent years, efforts have been made to develop the second generation reversible BTK inhibitors that are effective against both wild-type and C481S mutated B-cell malignancies. LOXO-305 and ARQ-531 are two examples of the second generation reversible BTK inhibitors in advanced clinical trials for ibrutinib-resistant CLL. Methods: New reversible BTK inhibitors were designed, synthesized and tested for enzymatic activities against wild-type and C481S-mutated BTK. Highly active compounds were confirmed for growth effects in diffuse large B-cell lymphoma derived TMD8 cells. Their microsomal stability and ADME properties were also assessed. Potent and bioavailable compounds were further evaluated in vivo efficacy using a TMD8 xenograft tumor model in mice. A 14-day toxicity study in rats was performed correspondingly. Results: As shown in the table, HBW-3-10 has greater potency and pharmacokinetic profile (rats, 10mg/kg PO) than ARQ-531. In a TMD8 mouse xenograft study, HBW-3-10, ARQ-531 and ibrutinib were compared directly, all dosed at 10mg/kg QD, and the resulting tumor growth inhibition rates (TGI) are 38.3%, 9.3% and 22.5%, respectively. Based on our data, HBW-3-10 is more efficacious than ARQ-531 and ibrutinib. In a 14-day preliminary toxicity experiment in rats, both HBW-3-10 and ARQ-531 were dosed 100mg/kg QD. Animals in ARQ531 group started showing signs of sickness on day 3, and all died on day 6 due toxicity; in contrast, no animal in HBW-3-10 group showed any sign of sickness or died during the entire course of study. Conclusions: We have discovered a novel second generation reversible BTK inhibitors HBW-3-10, that has a superior preclinical profile, efficacy and safety than other known ones. HBW-3-10 provides a valuable clinical candidate for treating Ibrutinib resistant CLL and beyond![Table: see text]

Single-Cell RNA-Seq Reveals LGALS1 and LAG3 as Novel Drivers of Ibrutinib Resistance in Chronic Lymphocytic Leukemia

Background: Chronic lymphocytic leukemia (CLL) is a highly heterogeneous malignant lymphoproliferative B-cell disorder that can be treated using ibrutinib, a Bruton’s tyrosine kinase inhibitor. However, the ibrutinib resistance of CLL patients has caused widespread concerns, necessitating the development of novel treatment strategies. Methods: Here, we identified lectin galactoside-binding soluble 1 (LGALS1) and lymphocyte-activating gene 3 (LAG3) as potential markers for ibrutinib-resistant CLL using single-cell RNA sequencing (scRNA-seq), and the results were validated in an ibrutinib-resistant CLL cell line (MEC1-IR) and primary cells from CLL patients. Marker-gene expression was detected while functional analyses were conducted with or without OTX008, a selective Galectin-1 inhibitor. ScRNA-seq revealed that the biological features, gene expression profiles, and clonal signatures of peripheral blood mononuclear cells (PBMCs) from patients with ibrutinib-resistant CLL were distinct from those displayed by PBMCs from ibrutinib-sensitive patients. Findings: A close correlation between LGALS1 and LAG3 expression was observed and these factors were found to be highly expressed in ibrutinib-resistant CLL, with diagnostic and prognostic stratification, indicating that they may serve as drivers of ibrutinib-resistant CLL. Concordantly, LGALS1 and LAG3 expression was higher in ibrutinib-resistant CLL cells and primary cells, and OTX008 suppressed proliferation and induced apoptosis in both cells. Interpretation: LGALS1 and LAG3 gene panel is promising indicator of ibrutinib-sensitivity and prognosis marker of CLL. LGALS1 inhibitor OTX008 is effective in CLL patients, both those naive to and those resistant to ibrutinib. This work highlights the pathogenic mechanism and represents a novel therapeutic target for CLL. Funding Statement: This work was supported by grants from National Natural Science Foundation of China (Grant #81720108002, 81970146, 81770166, 81972358 and 91959113), Key Research & Development Program of Jiangsu Province (Grant #BE2017733), Basic Research Program of Jiangsu Province (Grant #BK20180036), Jiangsu Province’s Medical Elite Programme (Grant #ZDRCA2016022), Project of National Key Clinical Specialty, Jiangsu Provincial Special Program of Medical Science (Grant #BE2017751), Six Top Talent Project of Jiangsu Province (Grant #WSN-001). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: Human sample study was approved by the Medical Ethics Committee of First Affiliated Hospital of Nanjing Medical University. Written informed consent was obtained from all participants.

Decreased NOTCH1 Activation Correlates with Response to Ibrutinib in Chronic Lymphocytic Leukemia.

Ibrutinib, a Bruton tyrosine kinase inhibitor (BTKi), has improved the outcomes of chronic lymphocytic leukemia (CLL), but primary resistance or relapse are issues of increasing significance. While the predominant mechanism of action of BTKi is the B-cell receptor (BCR) blockade, many off-target effects are unknown. We investigated potential interactions between BCR pathway and NOTCH1 activity in ibrutinib-treated CLL to identify new mechanisms of therapy resistance and markers to monitor disease response. NOTCH activations was evaluated either in vitro and ex vivo in CLL samples after ibrutinib treatment by Western blotting. Confocal proximity ligation assay (PLA) experiments and analyses of down-targets of NOTCH1 by qRT-PCR were used to investigate the cross-talk between BTK and NOTCH1. In vitro ibrutinib treatment of CLL significantly reduced activated NOTCH1/2 and induced dephosphorylation of eIF4E, a NOTCH target in CLL. BCR stimulation increased the expression of activated NOTCH1 that accumulated in the nucleus leading to HES1, DTX1, and c-MYC transcription. Results of in situ PLA experiments revealed the presence of NOTCH1-ICD/BTK complexes, whose number was reduced after ibrutinib treatment. In ibrutinib-treated CLL patients, leukemic cells showed NOTCH1 activity downregulation that deepened over time. The NOTCH1 signaling was restored at relapse and remained activated in ibrutinib-resistant CLL cells. We demonstrated a strong clinical activity of ibrutinib in a real-life context. The ibrutinib clinical efficacy was associated with NOTCH1 activity downregulation that deepened over time. Our data point to NOTCH1 as a new molecular partner in BCR signaling with potential to further improve CLL-targeted treatments.

Ibrutinib in the treatment of chronic lymphocytic leukemia: 5 years on.

A major revolution in the treatment of chronic lymphocytic leukemia (CLL) began with the approval of ibrutinib, a first-in-class oral inhibitor of Bruton tyrosine kinase (BTK), for the treatment of relapsed/refractory (R/R) and/or TP53 mutated patients with CLL. However, 5 years later, some issues relating to this disorder still remain including the fact that with ibrutinib only a relatively small proportion of patients achieve complete remission and that ibrutinib-resistant CLL clones can develop in about 20% of patients. In addition, therapy must still be given continuously, and toxicities leading to drug discontinuation occur in about 30% of patients. In the meantime second-generation BTK inhibitors have already aroused considerable interest and gathered momentum. A possible strategy to overcome some of these obstacles is to combine ibrutinib with other targeted agents especially in high-risk disease, such as previously treated refractory patients or those with TP53 aberrations or complex karyotypes, in whom rapid eradication of disease is most desirable. Therapy with single agent ibrutinib should be part of a sequential approach for patients with low risk disease, especially in older patients (aged >70 years) with a higher burden of comorbidities. Long-term results of ongoing studies combining Ibrutinib with (chemo)-immunotherapy or other targeted agents are eagerly awaited. Future clinical trials are indeed still needed to provide answers to these open questions.

Highly Potent BTK Degradation Induced By NRX0492 As a Therapeutic Strategy for CLL

B-cell receptor (BCR) signaling plays an essential role in normal B-cell development and is a key driver of proliferation and survival of chronic lymphocytic leukemia (CLL) B cells. BCR signals through BTK to NF-ĸB. Targeted inhibition of BTK with ibrutinib, a first-in-class covalent BTK inhibitor, is highly effective in treating CLL and related B-cell malignancies. However, progressive disease on continuous therapy with ibrutinib has been associated with mutations in BTK and PLCG2. Most patients with acquired resistance and progressive CLL on ibrutinib have mutations in BTK affecting the cysteine at position 481 (C481) to which ibrutinib covalently binds. The common C481S mutation increases IC50 with ibrutinib from low nM to μM concentrations. The emergence of specific BTK mutations in ibrutinib resistant CLL further validate BTK as a valuable therapeutic target in CLL. Therefore, agents that can target C481 mutant BTK are of great interest, and non-covalent BTK inhibitors have entered clinical trials. Here we investigated an alternative approach using a chimeric targeting molecule: NRX0492. NRX0492 combines two molecules: a "hook" that binds BTK with a "harness" that recruits ubiquitin ligases that mediate proteasome-dependent degradation of BTK. In cell line models, NRX0492 induce both wild-type and C481S mutant BTK degradation efficiently and specifically. Here we sought to define the on-target effects of NRX0492 in primary CLL cells in vitro and in vivo and to test NRX0492 against ibrutinib-resistant primary CLL cells. CLL cells were treated at densities of 5 × 106 cells/ml in 24-well plates with NRX0492, ibrutinib, the "hook" moiety devoid of the ubiquitin ligase binding moiety, or DMSO. BTK levels were quantified by Western blotting and flow cytometry. The mean ED50 and ED90 of NRX0492 for BTK degradation at 4 hours was 0.18nM and 0.5nM, respectively. At ED90 concentrations, mean cell viability of cultures treated with NRX0492 was 97.5%, and not different from viability of controls. Incubation at ED50 up to 24 hours also did not induce cell death. A lack of substantial cytotoxic activity with BTK degradation is in line with observations with BTK inhibitors under similar conditions. BTK degradation was achieved rapidly; in time-course experiments of CLL cells treated with 0.2nM NRX0492 the mean half-live of BTK protein was 2.7 hours (range 2.4h-3h), and at 2nM 99% of BTK was degraded within 4 hours. Next, we tested the rate of recovery of BTK expression after drug washout. After 0.2nm NRX0492 treatment for 4 hours, mean BTK levels were 55% of pre-treatment. After washout and continued culture of cells in complete medium for 96 hours, BTK levels were 41% of pre-treatment. In additional experiments with washout of NRX0492 after 4 hours, we found the lowest levels of BTK 48hours post-treatment, indicating a sustained drug effect. There was no significant difference in the efficacy of BTK degradation between CLL samples subdivided by IGHV mutation status or FISH categories. At 0.2nM NRX0492 for 4 hours, mean remaining BTK was 54% in IGHV mutated and 55% in IGHV unmutated samples (P =0.78), and 60% vs 50% for del(13q) vs del(17p) (P =0.18). Next, we studied samples from two patients progressing on ibrutinib with classic C481S mutations at cancer cell fractions of 50% and 84%, respectively. Both patients remained on ibrutinib at the time of sample collection. After 4 hours of treatment with 0.2nM NRX0492, BTK levels were 50% and 15% of DMSO-treated controls, respectively, consistent with degradation of mutant but not ibrutinib bound BTK. Wild-type BTK bound by ibrutinib is not degraded because ibrutinib prevents NRX0492 binding to BTK. In conclusion, we find NRX0492 is highly effective in degrading BTK in CLL cells. NRX0492 is effective at sub nM concentrations with ED50 ~0.2nM and ED90 ~0.5nM in primary CLL cells. Onset of BTK degradation was rapid and sustained for days after drug washout. NRX0492 was effective across CLL risk groups and equally effective in degrading wild-type BTK and C481S-mutant BTK. Studies testing NRX0492 in vivo in PDX models are ongoing and will inform dosing regimens for clinical studies. Disclosures Kelly: Nurix Therapeutics: Employment. Robbins:Nurix Therapeutics: Employment. Tan:Nurix Therapeutics: Employment. Ingallinera:Nurix Therapeutics: Employment. Sands:Nurix Therapeutics: Employment. Baskar:NIH: Patents &amp; Royalties: ROR1 mAb 2A2. Wiestner:Pharmayclics: Research Funding; Acerta: Research Funding; Merck: Research Funding; Nurix: Research Funding.

LOXO-305: Targeting C481S Bruton Tyrosine Kinase in Patients with Ibrutinib-Resistant CLL

Clinical success with the targeted Bruton Tyrosine Kinase (BTK) inhibitors ibrutinib and acalabrutinib has greatly improved the outcome of patients with relapsed chronic lymphocytic leukemia (CLL). However toxic side effects and acquired resistance due to C481S mutations in BTK remain an issue and the prognosis of those developing resistance is poor. Hence better therapeutic options are needed for these patients. Here we characterize a next generation highly selective reversible BTK inhibitor, LOXO-305, and describe its effectiveness in vitro in treatment naïve CLL patients and in those with C481S mutations. Unlike the irreversible BTK inhibitors ibrutinib and acalabrutinib, LOXO-305 does not require the C481 site for binding to the ATP binding domain of BTK. In addition, LOXO-305 is highly selective, with minimal activity against non-BTK kinase and non-kinase off targets, including ITK, TEC and EGFR. We evaluated the efficacy of LOXO-305 in BTK wild-type B cell lymphoma lines most similar to CLL, namely MEC1 and OSU-CLL, and in CLL patient samples, by determining its effect on cell viability, apoptosis and BCR signaling. In both cell lines LOXO-305 reduced cell viability and BCR mediated activation of BTK, PLCg2, ERK and AKT similar to ibrutinib. We measured cellular apoptosis in these cell lines with Annexin V APC and PI staining and showed that after 48 hours LOXO-305 treated cells had a significantly higher percentage of apoptotic cells in comparison with both DMSO (mean increase with LOXO-305: MEC1 21% (p = 0.0005) &amp; OSU-CLL 35% (p = 0.0016)) and ibrutinib (mean increase with LOXO305: MEC1 19% (p=0.0006), OSU-CLL 22% (p=0.0013)). Since LOXO-305's mechanism of BTK inhibition does not involve covalent binding to the C481 site, we tested its efficacy in in vitro ibrutinib resistant models of stably transfected HEK293 cells expressing either wild-type (WT) or C481S BTK. In WT BTK HEK cells, both LOXO-305 and ibrutinib showed comparable inhibitory activity in vitro against wild-type BTK, with IC50 values for phospho-BTK inhibition equal to 5.69 nM and 3.33 nM, respectively. LOXO-305 also inhibited phospho-BTK in BTK-C481S-expressing HEK293 cells at an IC50 equal to 21.2 nM, while ibrutinib had no inhibitory effect at concentrations as high as 300nM. When we extended our analysis to treatment naïve CLL patient cells, we observed that both LOXO-305 and ibrutinib potently inhibited IgM-induced phospho-BTK with IC50 values equal to 1.34 ± 1.23 nM for LOXO-305 (n = 7, p &amp;lt; 0.0001) and 1.04 ± 1.26 nM for ibrutinib (n = 7, p &amp;lt; 0.0001). We also found a significant reduction in phosphorylation of PLCγ2 (Y1217), the immediate downstream effector of BTK (IC50 33 nM for each agent, n = 7, p = 0.02 for LOXO-305, p = 0.0017 for ibrutinib). When we compared the cellular cytotoxicity of LOXO-305 with ibrutinib and acalabrutinib, all three agents demonstrated significant induction of apoptosis (annexin V positive) in BTK wild-type cells compared to DMSO treatment: (LOXO-305: median 63.77% n=3, p = 0.0073, ibrutinib: median 57.76%, n=3, p &amp;lt; 0.0001, acalabrutinib: median 67.11%, n=3, p = 0.0397). In CLL patient cells harboring C481S mutations, we observed a decrease in BCR signaling with 0.6 μM LOXO-305 treatment, with 95% reduction in phospho-BTK (Y223) and 50% reduction in phospho-PLCγ2 vs DMSO (n = 3, phospho-BTK p &amp;lt; 0.0001, phospho-PLCγ2 p = 0.01). In comparison ibrutinib at 0.6 μM demonstrated 50% reduction in phospho-BTK (n = 3, p = 0.01) and no significant change in phospho-PLCγ2. We also observed a 73% reduction in phospho-ERK vs DMSO with 10 μM LOXO-305 treatment (n=3, p = 0.004) while ibrutinib and acalabrutinib showed no significant change. In CLL patient cells with C481S variant allele frequencies (VAF) of 87% and 89%, LOXO-305 was 40-fold more potent at inhibiting phospho-BTK than ibrutinib (IC50 for LOXO305 0.02 μM, for ibrutinib 0.9 μM). Even in patient cells with lower C481S VAF of 9%, LOXO-305 still demonstrated 30-fold higher potency than ibrutinib (IC50 LOXO-305 0.2 μM, ibrutinib 0.6 μM). Our findings show that LOXO-305 potently inhibits cell survival and BCR signaling in both treatment naïve and C481S CLL patient cells, and therefore may prove an effective treatment in both treatment naïve and ibrutinib-resistant CLL patients. A phase 1 clinical trial is ongoing. Disclosures Ebata: LOXO Oncology Inc.: Employment, Equity Ownership. Gomez:LOXO Oncology Inc.: Employment, Equity Ownership. Brandhuber:LOXO Oncology Inc.: Employment, Equity Ownership. Rothenberg:LOXO Oncology Inc.: Employment. Brown:Janssen, Teva Pharmaceuticals: Honoraria; Gilead, Loxo, Sun Pharmaceuticals, Verastem: Research Funding; Morphosys, Invectys (Data Safety Monitoring Board): Membership on an entity's Board of Directors or advisory committees; Abbvie, Acerta, Astra-Zeneca, Beigene, Catapult Therapeutics, Dynamo Therapeutics, Genentech/Roche, Gilead, Juno/Celgene, Kite, Loxo, Octapharma, Novartis, Pfizer, Pharmacyclics, Sunesis, TG Therapeutics, Verastem: Consultancy.

Dual Inhibition of PI3K-δ and PI3K-γ By Duvelisib Eliminates CLL B Cells, Impairs CLL-Supporting Cells, and Overcomes Ibrutinib Resistance in a Patient-Derived Xenograft Model

Dual Inhibition of PI3K-δ and PI3K-γ By Duvelisib Eliminates CLL B Cells, Impairs CLL-Supporting Cells, and Overcomes Ibrutinib Resistance in a Patient-Derived Xenograft Model

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

AbstractThe 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.