IPI-145 antagonizes intrinsic and extrinsic survival signals in chronic lymphocytic leukemia cells

Chronic lymphocytic leukemia (CLL) cells display upregulated B-cell receptor (BCR) activation,^1,2  which is integral for maintaining B-cell survival and proliferation through transmitting microenvironmental stimuli.¹  Because of aberrant regulation of the BCR, CLL cells exhibit constitutively activated protein kinases, such as phosphoinositide-3 kinase (PI3K) and Bruton’s tyrosine kinase (BTK).^3,4  Small molecules that target such kinases, such as idelalisib⁵  and ibrutinib,⁶  have shown promising activity in CLL patients. PI3Ks are enzymes that integrate extracellular stimuli from membrane receptors by generating phosphatidylinositol 3,4,5-trisphosphate which serves as a critical plasma membrane docking site for pleckstrin-homology domain containing proteins, including AKT and BTK. Class I PI3K has 4 catalytic isoforms: p110α, p110β, p110δ, and p110γ. Although p110α and p110β are ubiquitously expressed, p110δ and p110γ are enriched in the hematopoietic system. Previously, we demonstrated that selective inhibition of p110δ with idelalisib antagonizes prosurvival signals of CLL cells.³  IPI-145 is an oral PI3K p110δ and p110γ inhibitor whose structure and activity in inflammatory disease models have previously been described.^7,8  It is well-tolerated and active in relapsed/refractory CLL patients and is currently being evaluated in the phase 3 setting as monotherapy for CLL (NCT02004522). Ibrutinib is an irreversible inhibitor of BTK that binds covalently to the cysteine residue (C481) in the kinase domain. It has been shown to be highly effective in CLL in various preclinical and clinical studies.^4,6  However, a small proportion of patients develop resistance.⁶  Our group has recently reported that resistant patients harbor a C481S mutation on the BTK gene that allows ibrutinib to reversibly bind to BTK.⁹  It is of interest to explore whether IPI-145 can inhibit prosurvival signaling through AKT in the setting of this C481S mutation. To better understand the mechanism of IPI-145 and whether it can overcome C481S ibrutinib resistance, we evaluated the activity of IPI-145 in various preclinical and ibrutinib-resistance models.

Blood was obtained from patients with CLL under the approval of the Institutional Review Board at The Ohio State University³  with informed consent in accordance with the Declaration of Helsinki. B, T, or natural killer (NK) were selected and maintained as previously described.³  The XLA cell line was obtained from the Coriell Institute. IPI-145 was supplied by Infinity Pharmaceuticals.

Immunoblots were performed as previously described.³  Antibodies included: anti-AKT, anti-phospho-AKT (ser473), anti-phospho-AKT (thr308), anti-ERK1/2, anti-phospho-ERK1/2 (thr202/tyr204), and anti-BTK from Cell Signaling Technologies; anti-phospho-BTK (tyr223) from Abcam; and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Millipore.

Cell viability was also measured using annexin-V/propidium iodide flow cytometry (Beckman-Coulter) as previously described.³ 

Th1/Th2/Th17 cytokines were measured using BD Cytometric Bead Array (BD Biosciences) according to the manufacturer’s published protocol.

The inducible Tet-on transactivator (Clontech) was stably introduced into the XLA cell line as previously described.¹⁰  The retroviral construct pRetroX-Tight-Puro was used to stably transfect XLA cells with BTK. A mutation was made using QuikChange site directed mutagenesis (Stratagene) in the kinase domain at cysteine 481 to serine (see the primer sequence in supplemental Methods on the Blood Web site). Confirmation of the DNA sequence, production of the viral particles, and infection of the XLA cell line were performed as previously described.¹¹  Cells were selected with puromycin and G418. BTK expression was induced with doxycycline for 48 hours.

To determine whether IPI-145 antagonizes BCR-crosslinking stimulation, we activated primary CLL cells using plate-bound anti-immunoglobulin M (IgM) and treated with IPI-145. Anti-IgM crosslinking resulted in the activation of pAKT^S473 in 4 of 6 patients samples tested. pAKT^S473 was significantly inhibited by IPI-145 at a concentration as low as 0.01 μM in 4 of 4 patients (Figure 1A). Moreover, IPI-145 completely abrogated the anti-IgM activated pAKT^T308 and downstream pERK^T202/Y204 signaling (Figure 1B). These data suggest that IPI-145 is effective at blocking PI3K activity and overcoming BCR-dependent microenvironmental protection.

The functional activity of IPI-145 was confirmed by viability analysis. Cell viability was measured every 24 hours (up to 72 hours); 0.25 to 5 μM IPI-145 exhibited concentration- (Figure 1C) and time-dependent (supplemental Figure 1) induction of cytotoxicity in CLL. This concentration range covers the plasma steady-state concentration (0.9 μM) and plasma peak concentration (C_max) (2.4 μM) for the patients who receive doses at 25 mg twice daily.¹²  The cytotoxic effect observed is independent of patients’ Ig heavy chain variable mutational status (supplemental Figure 2).

To examine the overall cytotoxicity of IPI-145 to immune cells, we incubated whole blood from CLL patients with 0.25 to 5 μM IPI-145 for 48 hours and analyzed it by flow cytometry for absolute count of live T, NK, and B cells. T and NK cells were sensitive to IPI-145; however the B-cell population was more sensitive (Figure 1D). To specifically examine normal B cells, we isolated B cells from healthy volunteer blood and incubated them with 1 μM IPI-145 for 48 hours and observed no cytotoxicity (Figure 1E), despite observing a significant decrease in CLL cells viability under the same conditions.

Previous studies have shown that PI3K p110δ, and p110γ are essential to cytokine production by immune effector cells. Although IPI-145 did not cause cytotoxicity to T cells (supplemental Figure 3), we sought to determine whether it affects the function of T cells. Upon CD3/CD28 stimulation, T cells showed significant increases in interleukin-1 (IL-2), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) production. IL-2 was inhibited by IPI-145 starting at 0.5 μM, and a trend in inhibition of TNF-α and IFN-γ was also observed (Figure 1F). Because these inflammatory cytokines are prosurvival to CLL cells, this inhibitory effect of IPI-145 could further enhance CLL cytotoxicity in vivo. However, these cytokines are also important for the normal inflammatory response, and the inhibitory effect could associate with opportunistic infection.

Finally, to understand whether IPI-145 can potentially overcome ibrutinib resistance, we established an in vitro ibrutinib resistant cell line model. BTK-null XLA cells were transfected with wild-type or C481S BTK.¹¹  Phosphorylation of wild-type BTK was inhibited by short-term ibrutinib treatment; however, phosphorylation of C481S mutant BTK could not be inhibited to the same extent. AKT phosphorylation in wild-type cells but not C481S BTK cells can be inhibited by ibrutinib. In contrast, IPI-145 completely abrogated AKT phosphorylation irrespective of BTK mutation status (Figure 2). We observed similar signaling blockade in accordance with cytotoxicity in primary CLL cells that were isolated from BTK C481S mutated ibrutinib resistant patients (supplemental Figure 4).

Here we provide data characterizing IPI-145 in CLL for the first time and as the first report describing the activity of IPI-145 in an ibrutinib resistance model. We demonstrated that IPI-145, an inhibitor of PI3K p110δ and p110γ, antagonized prosurvival signaling in BCR cross-linked CLL cells and promoted apoptosis in a dose- and time-dependent fashion, whereas not generating direct cytotoxicity to normal B cells. These apoptosis results are similar to those previously reported by us with idelalisib.³  IPI-145 also reduces the viability of normal T and NK cells and decreases activated T-cell production of various inflammatory and antiapoptotic cytokines. Moreover, we proved that IPI-145 is effective at reducing downstream PI3K signaling as evidenced by diminished AKT phosphorylation even in the setting of BTK C481S mutation. This study provides solid rationale for ongoing clinical development of IPI-145 in CLL patients. Additionally, this study provides justification for clinical use of IPI-145 in the subset of patients that harbor C481S BTK mutation, most likely in combination, given the potential of this subset of CLL patients to develop resistance to monotherapy kinase inhibitors.

The authors thank the families who provided samples for this work.

This work was supported by the Specialized Center of Research from the Leukemia and Lymphoma Society, the National Institutes of Health National Cancer Institute (K12 CA133250, P50-CA140158, P01 CA95426, P01 CA81534, and P01 CA101956), and the D. Warren Brown Foundation, the Four Winds Foundation, the Sullivan Chronic Lymphocytic Leukemia Research Fund, Mr and Mrs Al Lipkin, Mr and Mrs Michael Thomas, and The Harry T. Mangurian Foundation.

Contribution: S.D., A.J.J., and J.C.B. designed the experiments, analyzed the data, wrote the article, and reviewed and approved the final version; D.G. established, characterized, and provided the ibrutinib resistance cell line model, reviewed and modified versions of the article, and approved the final version; and J.A.D., Y.Z., A.L., J.K., and J.A.W. planned and contributed to components of the experimental work presented, reviewed and modified versions of the article, and approved the final version.

Conflict-of-interest disclosure: J.K. is an employee of Infinity Pharmaceuticals. The remaining authors declare no competing financial interests.

Correspondence: Amy J. Johnson, Ohio State University Comprehensive Cancer Center Bldg. Room 455C, 410 West 12th Avenue, Columbus, OH 43210; e-mail: amy.johnson@osumc.edu.