Ibrutinib Disrupts IL-4R - IL-4 Axis By Inhibiting IL-4R Signaling and Reversing Th2/Th1 Polarization through Diminished CLECL1 in CLL B Cells

The BTK inhibitor, ibrutinib, inhibits chronic lymphocytic leukemia (CLL) cell growth by blocking microenvironmental support in situ, releasing cells from tissues niches, and diminishing IL-4-producing T cells, all of which deprive leukemic cells of nurturing signals. However, little is known about the mechanisms whereby ibrutinib affects surface membrane (sm) levels of IL-4R and IL-4R signaling, Th2 polarization, particularly the relative sensitivities of U-CLL versus M-CLL to these effects of the drug, and how these parameters impact on those intraclonal CLL B cell fractions defined by time since last cell division. Hence we addressed these issues here.

We studied 28 treatment-naive CLL patients receiving ibrutinib as single agent therapy. CLL B cells collected after ibrutinib treatment expressed lower densities of sm CD5, CD20, HLA-DR, CD25, and CD28; they also transiently displayed increased smIgM levels and persistently exhibited elevated but signaling-impeded smCXCR4. Moreover, ibrutinib treatment significantly lowered smIL-4R, without changing RNA or total protein content of the residual cells, suggesting that ibrutinib treatment may interrupt membrane trafficking. Finally, CLL B cells treated with ibrutinib exhibited significantly lower pSTAT6 and pJAK3 upon IL-4 stimulation, a here-to-fore unknown effect of BTK inhibition. Thus, ibrutinib impairs IL-4R sm expression and function.

We next investigated if ibrutinib-impaired IL-4R signaling differentially affected the survival of U-CLL versus M-CLL cells. Supplementing leukemic B cell cultures with autologous T cells improved survival of U-CLL but not M-CLL cells. This T cell promoted survival was blocked by pretreatment of T cells with ibrutinib, or by co-culturing cells with IL-4 neutralizing antibody. Similarly, IL-4 improved the survival of U-CLL but not M-CLL cells in vitro, but only if the CLL B cells were tested before, but not after ibrutinib treatment in vivo . These findings were confirmed in a xenograft mouse model using primary treatment-naïve CLL patient cells; we observed greater inhibition of tissue-resident CLL B cell growth for U-CLL than M-CLL with ibrutinib therapy. Thus, IL-4R signaling is critical for CLL B cell survival, and U-CLL cells are more dependent on this than M-CLL.

The CLL B cells remaining post-ibrutinib treatment were smaller, less viable and exhibited the resting phenotype described above; in almost all cases, ibrutinib eliminated the recently born, "proliferative fraction" (PF) of CLL cells, defined by a CXCR4^DimCD5^Bright phenotype. As suggested by our gene expression studies, we found that CLL PF cells display the highest levels of smIL-4R and smCLECL1, the latter is a receptor that induces Th2 polarization. Here we documented this by T cell polarization assays using autologous naïve CD4+ T cells co-cultured with CLL PF cells. However after ibrutinib treatment, PF cells taken from patients expressed lower smCLECL1 and failed to induce Th2 polarity. Thus, the Th1 polarization bias that occurs with ibrutinib treatment might result from direct targeting of ITK in T cells, as suggested by others, and by indirect targeting of CLECL1 on CLL cells as demonstrated here.

Finally, PF cells from U-CLL patients have the highest levels of the anti-apoptotic protein BCL2. Ibrutinib treatment for 1 cycle significantly reduced BCL2 protein in U-CLL but not M-CLL cases. Notably, U-CLL cases with initial reduction of BCL2 that we followed to the end of cycle 3 treatment displayed increased BCL2 after cycles 2 and 3, suggesting that BTK inhibition initially selects clones expressing higher levels of BCL2. M-CLL cases however had invariant BCL2 protein during treatment, which might contribute to the better survival of M-CLL cells after the loss of environmental support.

In summary, we report that ibrutinib interrupts the IL-4R - IL-4 axis by diminishing smIL-4R, smCLECL1, intracellular BCL2; and by promoting a non-IL-4 microenvironment thus favoring a Th1 cell bias. U-CLL B cells, in particular their PF, are more vulnerable to this action than M-CLL cells and therefore are more prone to die in tissue niches, consistent with M-CLL patients having greater and longer lymphocytosis when treated with ibrutinib. Thus our results provide a mechanistic understanding for the differences in primary ibrutinib sensitivity between U-CLL and M-CLL patients, supporting the rationale to combine inhibitors of BTK and BCL2 in CLL.