CLL Cell Viability Promoted by Myosin Heavy Chain IIA Exposed Apoptotic Cells is BTK-dependent
Authors: Chu Charles C. , Zhang Lu , Cui Xiaoxuan , Magli Amanda R. , Yan Xiao-Jie , Kolitz Jonathan E. , Allen Steven L. , Barrientos Jacqueline C. , Rai Kanti R. , Chiorazzi Nicholas 
Published: October 2019

Abstract
Abstract 1767
B-cell chronic lymphocytic leukemia (CLL) is a clonal CD5+CD19+ B-lymphocyte cancer with a B cell receptor (BCR) immunoglobulin heavy chain variable (IGHV) gene sequence that may be classified as unmutated (U-CLL) or mutated (M-CLL) depending on the level of IGHV mutation. Because U-CLL patients have a worse clinical prognosis than M-CLL patients, the structure of the BCR and the signaling consequences of BCR occupancy by antigen are considered crucial to disease development and possibly progression. Moreover, nearly one third of CLL patients express a CLL BCR that is virtually identical in sequence (stereotyped) to other CLL patient subgroups, an incidence that is extraordinarily unlikely by random chance, suggesting that common antigens could bind to these CLL BCRs and initiate signaling. Apoptotic autoantigens may be a source of such antigens. Indeed, CLL BCRs expressed as native or recombinant monoclonal antibodies (mAbs) can recognize autoantigens, such as nonmuscle myosin heavy chain IIA (myosin), actin, vimentin, cofilin-1, filamin B, insulin, DNA, IgG, myoglobin, thyroglobulin, cardiolipin, and oxidized low-density lipoprotein. These molecules are generally intracellular and may be exposed on the cell surface during apoptosis, permitting binding to CLL BCRs. Indeed, intracellular myosin is exposed on the surface of a subset of apoptotic cells, termed MEACs (myosin exposed apoptotic cells). Furthermore, MEACs bind to over 60% of CLL mAbs tested, supporting the idea that multiple autoantigens exposed on MEACs may stimulate CLL cells. This binding may furnish signals beneficial to the leukemic cell, because MEAC binding correlates with shorter patient survival.
To test this hypothesis, the effect of MEAC binding on spontaneous CLL cell apoptosis was tested after co-culture of CLL peripheral blood mononuclear cells from a large patient cohort with MEACs (3:1–5:1 ratio) for 2–4 days using flow cytometry and AnnexinV and 7-actinomycin D staining to measure apoptosis in CD19+CD3− cells. A typical wide range of spontaneous apoptosis was observed (median = 26.98% (5.93%–91.35%) AnnexinV+). In this large patient cohort (n=64, with some patient samples tested 2–8 times), co-culture of CLL cells with MEACs resulted in a decrease in apoptosis in all patient samples as compared to cultures without MEACs (median = 18.58% (2.49%–76.35%) AnnexinV+), a highly significant result (P=0.0001, two-tailed paired t-test). In this cohort, U-CLL patients (n=23) had a slightly higher median decrease in apoptosis (30.39%) than M-CLL patients (n=33, 22.69%), but this was not significant (P=0.3940, two-tailed unpaired t-test with Welch's correction). Furthermore, no apparent correlation between MEAC co-culture responsiveness and in vitro MEAC binding was observed in the 9 CLL patient samples exhibiting stereotyped CLL BCR. Finding that MEAC co-culture affects all CLL cells regardless of IGHV mutation status or stereotypy contrasts with MEAC binding to CLL mAbs in vitro, for which there is a marked preference for U-CLL and stereotyped mAbs. One possible explanation for the conflicting result is that CLL cells with BCRs that do not bind MEACs well receive an additional non-Ig receptor signal that boosts the BCR signal to make it near equivalent to the signal from BCRs on cells that bind MEACs well. In this model, inhibition of BCR signaling would abrogate the decrease in apoptosis produced by MEAC co-culture in all CLL cases. To test this, Bruton tyrosine kinase (BTK) inhibitors, ibrutinib or LFM-A13, were added to these co-cultures (n=12 and 18, respectively). Both inhibitors significantly abrogated the MEAC co-culture increase in viability (ibrutinib, P=0.0005; LFM-A13, P=0.0007; two-tailed paired t-test). Ibrutinib and LFM-A13 inhibited all samples regardless of IGHV mutation status (stereotyped BCR samples were not tested).
In conclusion, these results are consistent with the theory that antigenic stimulation of CLL cells via the BCR promotes CLL cell viability and may influence the level of disease activity. MEACs may be a source of such antigenic stimulation. Interference with BCR signaling via BTK inhibition prevents this stimulation and may be one of the explanations for the clinical success of BCR signaling inhibitors in treating CLL.

Disclosures:
Barrientos: Gilead and Pharmacyclics: Research Funding.