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. 2020 Oct 27;4(20):5093-5106.
doi: 10.1182/bloodadvances.2019001369.

IBL-202 is synergistic with venetoclax in CLL under in vitro conditions that mimic the tumor microenvironment

Yandong Shen  1   2 , Kyle Crassini  1 , Narjis Fatima  1   2 , Michael O'Dwyer  3 , Michael O'Neill  4 , Richard I Christopherson  2 , Stephen P Mulligan  1   2 , O Giles Best  1   2   5
Affiliations

IBL-202 is synergistic with venetoclax in CLL under in vitro conditions that mimic the tumor microenvironment

Yandong Shen et al. Blood Adv. .

Abstract

The B-cell receptor signaling pathway and dysregulation of the Bcl-2 family of proteins play crucial roles in the pathogenesis of chronic lymphocytic leukemia (CLL). Despite significant advances in the treatment of the disease, relapse and drug resistance are not uncommon. In the current study, we investigated the dual PI3/PIM kinase inhibitor IBL-202 in combination with venetoclax as a treatment option for CLL using both primary CLL cells and TP53-deficient OSU-CLL cells generated using the CRISPR-Cas9 system. IBL-202 and venetoclax were highly synergistic against primary CLL cells cocultured with CD40L fibroblasts (combination index [CI], 0.4, at a fractional effect of 0.9) and TP53-knockout (KO) OSU-CLL cells (CI, 0.5, at a fractional effect of 0.9). Synergy between the drugs was consistent, with a significant (P < .05) reduction in the 50% inhibitory concentration for both drugs. IBL-202 and venetoclax in combination induced cell-cycle arrest and slowed the proliferation of both wild-type and TP53-KO cell lines. The drug combination inhibited AKT phosphorylation, reduced expression of Bcl-xL and NF-κB, and increased the Noxa/Mcl-1 ratio. Downregulation of CXCR4 was consistent with inhibition of the SDF-1α-induced migratory capacity of CLL cells. Synergy between IBL-202 and venetoclax against primary CLL cells cultured under conditions that mimic the tumor microenvironment suggests this drug combination may be effective against CLL cells within the lymph nodes and bone marrow. Furthermore, the efficacy of the combination against the TP53-KO OSU-CLL cell line suggests the combination may be a highly effective treatment strategy for high-risk CLL.

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Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
IBL-202 and venetoclax are synergistic against CLL cells. (A) Dose responses for IBL-202 and venetoclax, alone and in combination, at a ratio of 1:100 against primary CLL cells cocultured with CD40L-expressing fibroblasts (n = 10) or HS-5 stromal cells (n = 6) as indicated. The viability of CLL cells was assessed by DilC1(5)/PI staining and flow cytometry after 48 hours of treatment. Flow cytometric data from 1 representative patient sample illustrates the viability of CLL cells cocultured with CD40L fibroblasts after treatment with IBL-202 and venetoclax at 0.5 µM and 5 nM, respectively, alone or in combination. Results are expressed relative to vehicle-treated control cultures. (B) CI plot (left) and isobologram (right) for IBL-202 and venetoclax in combination against CLL patient samples (n = 10) cocultured with CD40L-expressing fibroblasts illustrates synergy between the 2 drugs. Data are presented as the CI over a range of fractional effects, where a fractional effect value of 0.5, for example, is indicative of 50% cell kill. (C) The proportion of viable CLL and T cells in mixed PBMC fractions from 4 CLL patients before and after drug treatment were determined by flow cytometry using antibodies to CD19 and CD5. PBMC fractions from CLL patients were treated with IBL-202 (1 μM) or venetoclax (10 nM) or the combination for 24 hours in medium. Representative plots from 1 patient are shown.
Figure 2.
Figure 2.
IBL-202 and venetoclax inhibit PI3K signaling and downregulate expression of Bcl-2 family proteins. Expression of phosphorylated and total AKT and Bcl-xL (n = 8) (A) and phosphorylated and total Bad and NF-κB and c-Myc (n = 6) (B) was assessed by immunoblotting in primary CLL cells cultured in medium alone or cocultured with CD40L fibroblasts. CLL cells in coculture with fibroblasts were treated with IBL-202 (1 μM) or venetoclax (10 nM), alone or in combination. Levels of Mcl-1, Noxa, Bax, Bcl-2, Bcl-xL, and PARP were assessed by immunoblotting of primary CLL cells treated with IBL-202 (1 μM) and venetoclax (10 nM), alone or in combination (n = 6) (C), and ibrutinib (0.5 μM) or venetoclax (10 nM), alone or in combination (n = 3) (D). Representative data from 2 patient samples are shown in panels A-D. The values shown under each lane indicate the fold change in expression relative to an untreated control for each patient sample. (E) Histograms show the cumulative data of the Noxa/Mcl-1 expression ratio in CLL cells treated with IBL-202 (n = 6) or ibrutinib (n = 3), alone or in combination with venetoclax. ns, not significant.
Figure 3.
Figure 3.
IBL-202 and venetoclax are synergistic against OSU-CLL-TP53ko cells. (A) Dose responses for IBL-202 or venetoclax as single agents toward wild-type (WT) OSU-CLL and OSU-CLL-TP53ko cells. (B) CI plot and isobolograms for IBL-202 and venetoclax in combination against WT and OSU-CLL-TP53ko cells. CI values <1 indicate synergy. (C) Cell-cycle analyses performed on control and OSU-CLL-TP53ko cells after treatment for 72 hours with the indicated dose of each drug. (D) Proliferation analysis of control and OSU-CLL-TP53ko cells treated with IBL-202 or venetoclax alone or in combination over a 72-hour time course. MFI, mean fluorescence intensity.
Figure 4.
Figure 4.
IBL-202 plus venetoclax decreases expression of CD49d and CXCR4 and impairs the migratory capacity of primary CLL cells. Changes in expression of CD49d and CXCR4 (A) and the migration of primary CLL cells down an SDF-1α gradient (n = 6) (B) were determined by flow cytometry after treatment with IBL-202 (1 μM) or venetoclax (10 nM) alone or in combination. Changes in antigen expression are presented using mean fluorescence intensity (MFI) values relative to levels in untreated cells. Flow cytometric plots from 1 representative patient sample illustrating the effects of IBL-202 and venetoclax, alone and in combination, on the number of viable cells migrating across a permeable support are shown. The number shown in the top left quadrant of each plot indicates the viable cell count under each condition.
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