Combined BTK and PI3Kδ Inhibition with Acalabrutinib and ACP-319 Improves Survival and Tumor Control in CLL Mouse Model

Abstract

Purpose: Targeting the B-cell receptor (BCR) pathway with inhibitors of Bruton tyrosine kinase (BTK) and PI3Kδ is highly effective for the treatment of chronic lymphocytic leukemia (CLL). However, deep remissions are uncommon, and drug resistance with single-agent therapy can occur. In vitro studies support the effectiveness of combing PI3Kδ and BTK inhibitors.Experimental Design: As CLL proliferation and survival depends on the microenvironment, we used murine models to assess the efficacy of the BTK inhibitor acalabrutinib combined with the PI3Kδ inhibitor ACP-319 in vivo We compared single-agent with combination therapy in TCL1-192 cell-injected mice, a model of aggressive CLL.Results: We found significantly larger reductions in tumor burden in the peripheral blood and spleen of combination-treated mice. Although single-agent therapy improved survival compared with control mice by a few days, combination therapy extended survival by over 2 weeks compared with either single agent. The combination reduced tumor proliferation, NF-κB signaling, and expression of BCL-xL and MCL-1 more potently than single-agent therapy.Conclusions: The combination of acalabrutinib and ACP-319 was superior to single-agent treatment in a murine CLL model, warranting further investigation of this combination in clinical studies. Clin Cancer Res; 23(19); 5814-23. ©2017 AACR.

Figure 1. Effects of inhibiting BTK, PI3Kδ or both on tumor burden, cell death and proliferation in mice carrying TCL1-192 cells

All data shown are at four weeks after TCL1-192 cell injection in NOD/SCID mice. Each experimental cohort consisted of 3-5 mice per treatment group. (a) Representative dot plots of CD5/B220 staining in mice receiving treatment as indicated. Percentage TCL1-192 cells (CD5⁺B220^hi) are indicated for each treatment. (b) Absolute numbers of TCL1-192 cells/μL in the peripheral blood of mice receiving treatment as indicated, measured as in panel a. Each symbol represents one mouse; different symbols represent different experimental cohorts. Line represents median. (c) Mean ± SEM absolute number of proliferating (KI67+) TCL1-192 cells in each treatment group, n=40 split evenly across treatment groups in two experimental cohorts. (d) Mean ± SEM absolute number of viable TCL1-192 cells in the peripheral blood for each treatment group, measured as Annexin-V and VIVID double negative cells, n=80 split evenly across treatment groups in four experimental cohorts. Abbreviations: Veh, vehicle; Acala, acalabrutinib; Combo, combination treatment with acalabrutinib and ACP-319. All comparisons by an unpaired t-test using a linear model to take into account the random batch effect: **P<0.01, ***P<0.001 and ****P<0.0001.

Figure 2. Impact of acalabrutinib, ACP-319 or their combination on tissue-resident TCL1-192 cells

TCL1-192 cells were harvested from the spleen four weeks after cell injection. (a–b) Each symbol represents one mouse; different symbols represent different experimental cohorts. (a) Occupancy of BTK in mice treated with either acalabrutinib alone or the combination of acalabrutinib and ACP-319. (b) Absolute number of recovered TCL1-192 cells harvested from the spleen as measured by flow cytometry, based on counting beads and the volume used for preparation of single cell suspension from the spleens. Line represents median. (c–d) Measurements were normalized to vehicle control. (c) Mean ± SEM percentage change in the proportion of KI67+ TCL1-192 cells in each treatment group compared with vehicle treated mice (n=20 split evenly across treatment groups) are shown. (d) Mean ± SEM percentage change in the proportion of viable TCL1-192 cells in each treatment group compared with vehicle treated mice (n=40 split evenly across treatment groups in two experimental cohorts). Measured as Annexin-V and VIVID double negative cells. (e) Spleen weights at time of sacrifice by treatment group. Each symbol corresponds to one mouse; the type of symbol/color represents the experimental cohort. Line represents median. (f) Spleens from representative mice in the different treatment groups. Abbreviations: Veh, vehicle; Acala, Acalabrutinib; Combo, combination treatment with acalabrutinib and ACP-319. All comparisons by an unpaired t-test using a linear model to take into account the random batch effect. Statistics comparing treatment to vehicle control are shown either above the treatment groups (panels b and f) or below the treatment bars (panels c and d); statistics comparing treatments are shown with comparison brackets: *P<0.05, ***P<0.001 and ****P<0.0001.

Figure 3. The combination of acalabrutinib and ACP-319 improves survival of mice injected with TCL1-192 cells compared with single-agents

Kaplan-Meier survival curves for mice injected with TCL1-192 cells and treated with either vehicle, acalabrutinib, ACP-319, or the combination of the two (n=40 split evenly across treatment groups in two experimental cohorts). Tx indicates start of treatment (15 days from cell injection). Abbreviations: Veh; vehicle, Acala; Acalabrutinib; Combo, combination treatment with acalabrutinib and ACP-319. Comparisons by log-rank test (Mantel-Cox); P<0.0001 for each treatment group vs vehicle and for combination treated mice vs each single-agent treated group.

Figure 4. Effects of inhibiting BTK, PI3Kδ or both on ERK and NF-κB signaling in TCL1-192 cells

Treatment was started two weeks after TCL1-192 cell injection and mice were sacrificed four weeks after cell injection. (a) Mean ± SEM absolute numbers of pERK+ TCL1-192 cells in the peripheral blood, n=40 split evenly across treatment groups in two experimental cohorts. (b) Mean ± SEM percentage change in the proportion of pERK+ TCL1-192 cells in the spleen of mice in each treatment group compared with vehicle treated mice, n=20 split evenly across groups. (c) Mean ± SEM absolute numbers of phospho-NF-κB+ (p65) TCL1-192 cells in the PB (n=40 split evenly across treatment groups in two experimental cohorts). (d) Mean ± SEM percentage change in the proportion of phospho-NF-κB+ TCL1-192 cells in each treatment group compared with vehicle treated mice in the spleen (n=20 split evenly across treatment groups). Abbreviations: Acala, Acalabrutinib; Combo, combination treatment with acalabrutinib and ACP-319. All comparisons by an unpaired t-test using a linear model to take into account the random batch effect. Statistics comparing treatment to vehicle control are shown either above the treatment groups (panels a and c) or below the treatment bars (panels b and d); statistics comparing treatments are shown with comparison brackets: *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.

Figure 5. Inhibition of anti-apoptotic mechanisms in spleen-resident TCL1-192 cells

TCL1-192 cells were harvested from the spleen four weeks after cell injection and two weeks after treatment start. Left panels show representative immunoblots stained for each protein of interest and the loading control, γ-tubulin. Bands were quantitated by densitometry and normalized to loading control. Right panels show the mean ± SEM change in normalized expression as compared with vehicle-treated mice. (a) IκBα, n=24 split across treatment groups in two experimental cohorts, (b) Bcl-xL, n=24 split across treatment groups in two experimental cohorts and (C) Mcl-1, n=16 split across treatment groups in two experimental cohorts. Abbreviations: Veh, vehicle, Acala, Acalabrutinib and Combo, combination treatment with acalabrutinib and ACP-319. All comparisons by an unpaired t-test using a linear model to take into account the random batch effect. Statistics comparing treatment to vehicle control are shown above (panel a) or below (panels b and c) the treatment bars; statistics comparing treatments are shown with comparison brackets: *P<0.05 and **P<0.01.