In Vivo Evidence of in Situ Cell Death Preferentially Occurring in CLL Patients with Minimal Lymphocytosis By the Diminished BCL2 and Loss of T Cell Support after Ibrutinib Treatment
Authors: Chen Shih-Shih , Ham Steven , Ravichandran Priyadarshini , D'Aloisio Ileana , Butera Emily Kate , Lyallpuri Romi , Kieso Yasmine , Barrientos Jacqueline C. , Rai Kanti R , Burger Jan , Chiorazzi Nicholas 
Published: October 2019

Abstract
Chronic lymphocytic leukemia (CLL) patients receiving the BTK inhibitor ibrutinib develop lymphocytosis. Prolonged lymphocytosis is associated with good prognosis in both treatment-naïve (TN) and relapsed/refractory (R/R) patients, although in general R/R cases have higher lymphocytosis peaks. Because the degree and duration of the elevations vary between patients, lymphocytosis is likely not caused solely by release of cells from tissue niches. Mathematical modeling suggests less profound lymphocytosis of shorter duration is due to loss of tissue resident cells in situ. To address contributions of local elimination and egress to the periphery, we performed phenotypic and functional analyses of cell migration, survival/growth and interactions of CLL B and T cells, and correlated these results with ibrutinib induced T and B cell lymphocytoses.
Studies of CLL B cell lymphocytosis were performed with TN (17 U- and 11 M-CLL) and R/R (5 U-CLL) cases receiving single-agent ibrutinib therapy. Faster resolution of lymphocytosis was seen in TN U-CLL than in M-CLL cases (P = 0.008). Conversely, all 5 R/R U-CLL patients exhibited considerably longer lymphocytosis upon ibrutinib use (median time to normalization in TN vs R/R U-CLL: 17 vs 77 days, P = 0.01). Nevertheless, after treatment, all cases showed reduced surface levels of chemokine receptors (CXCR5, CXCR7, CCR7), integrins (CD11a, CD49d) and elevated numbers of CXCR4 molecules that were non-functional based on impaired receptor signaling and recycling/internalization. Also, CLL B cells collected after 2 cycles of ibrutinib treatment exhibited impaired chemotaxis to lymphoid tissues in alymphoid NSG mice. However, none of these changes differed between CLL cases with minimal or prolonged lymphocytosis.
We next investigated ibrutinib induced changes in tissue resident cells in a xenograft mouse model. Activated autologous T cells and PBMC prepared from TN (2 U- and 3 M-CLL) and R/R (1 U-CLL) patients were injected into NSG mice. 14 days later, mice were given ibrutinib daily for 2 weeks. Ibrutinib significantly inhibited CLL B cell growth in spleens in all 5 TN cases but to a much greater extent in U-CLL than M-CLL (P = 0.01). The R/R case also had reduced CLL B cell numbers in spleens after treatment, but this was not statistically significant. Molecules controlling CLL B cell survival were then examined in 15 TN and 5 R/R cases. Ibrutinib reduced BCL2 protein levels in TN patients with minimal lymphocytosis but not those with prolonged lymphocytosis (P < 0.001) or R/R patients (P = 0.017). These data suggest those tissue resident cells that are lost after ibrutinib therapy have reduced BCL2 protein levels.
In CLL T cells, ibrutinib targets ITK, an enzyme involved in T cell migration and function. Indeed, we found ibrutinib induced T cell lymphocytosis in the same cohort of patients described above. Shorter T cell lymphocytosis was seen in TN U- but not M-CLL or R/R cases (19.6±4 vs 44.3±13, P = 0.04). Ibrutinib blocked T cell chemotaxis in all the cases, but only significantly inhibited T cell growth of TN U-CLL cases in the same xenograft studies. The loss of T cells correlates with the greater inhibition of CLL B cells observed in TN U-CLL rather than in M-CLL cases. Supporting this finding, patients with minimal B cell lymphocytosis also had rapid resolution of T cell lymphocytosis. These data indicate ibrutinib blocked T cell growth in situ, especially in TN U-CLL patients. The loss of T cells further enhances CLL B cell death in tissues after ibrutinib treatment as autologous T cells can protect U-CLL cells from apoptosis by upregulating BCL2.
Collectively, our findings suggest that although ibrutinib promotes T and B cell egress in almost all the CLL cases, patients with prolonged lymphocytosis after ibrutinib treatment have better cell survival in situ even after loss of tumor microenvironment contact. In situ cell death after ibrutinib treatment preferentially occurs in TN U-CLL but not in TN M-CLL and R/R cases, in part due to loss of BCL2 and of the support from non-neoplastic immune cells like T cells, documenting that these beneficial actions of ibrutinib are intimately involved in the numbers of lymphocytes that circulate after drug administration. Finally, our data are consistent with the previous report that ibrutinib increases leukemic cell sensitivity to venetoclax, providing a strong rationale for combination therapy of ibrutinib and venetoclax in CLL.

Disclosures
Barrientos: Janssen: Consultancy; Gilead: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding. Burger:Gilead: Research Funding; Roche: Other: Travel, Accommodations, Expenses; Janssen: Consultancy, Other: Travel, Accommodations, Expenses; Portola: Consultancy; Pharmacyclics, LLC, an AbbVie Company: Research Funding.