Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in Europe and North America. A key feature of CLL is its extremely variable clinical outcome. Diverse genetic and epigenetic lesions, different phenotype profile and functional status of signaling molecules in malignant CLL B cells are molecular underpinnings of disease heterogeneity. This thesis is devoted to the characterization of CD150 isoforms expression, topology and signaling properties in CLL B cells. Immunophenotyping of CLL cases showed significant heterogeneity in expression of CD150, CD180, CD20, CD22 and CD95 expression. CD150 cell surface expression (csCD150) was detected in 71.6% of examined CLL cases with the portion of positive cells in the range from 7 to 93%. In 50.8% CLL cases B cells expressed low levels of csCD150. Medium expression level of CD150 was detected in 10.4% of CLL cases. Only 10.4% of CLL cases were characterized by high level of CD150 cell surface expression with more than 60% CD150+ CLL B cells. The expression of csCD150 positively correlated with the expression of the CD37, CD180, CD95, CD48, and CD5. It should be noted that in 59% of CLL cases B cells coexpress CD150 and CD180. Moreover, CD180 showed the highest level of colocalization with CD150 on the cell surface of CLL B cells. In the absence of CD150 and CD180 on the cell surface both receptors were expressed in the cytoplasm. The CD150 receptor was colocalized with markers of the endoplasmic reticulum, the Golgi apparatus and early endosomes. In contrast, CD180 was detected preferentially in early endosomes.
To find out which CD150 isoforms are expressed in CLL B cells, we focused on the canonical transmembrane CD150 isoform (mCD150) that has two ITSM signaling motifs in cytoplasmic domain, the secreted CD150 isoform (sCD150) that lacks the transmembrane region, and a novel CD150 isoform (nCD150) with an alternative cytoplasmic tail without known signaling motifs. To discriminate the mRNA expression of these CD150 isoforms we performed qRT-PCR using primers that are specific to unique region of each isoform. The mRNA expression level of the mCD150 isoform positively correlated with CD150 cell surface levels (r=0.4, p<0.05) in CLL B cells with the median of expression in the range of normal B cell subsets. We showed that regardless of CD150 cell surface expression the mCD150 was a predominant CD150 isoform in 85% of CLL cases. However, 15% of studied CLL cases were characterized by elevated nCD150 mRNA levels. The majority of CLL cases had significantly increased expression level of the soluble sCD150, moreover CLL B cells secrete this isoform.
To find out whether csCD150- and csCD150+ CLL B cells are different in their signaling network profiles we used two approaches: (I) comparison of signaling profiles of csCD150- and csCD150+ CLLs and (II) testing whether CD150 ligation trigger signaling events in CLL B cells. The csCD150- and csCD150+ CLL B cells had different patterns of constitutive phosphorylation. In the csCD150+ CLL samples the basal levels of tyrosine phosphorylation and phosphorylation of serine/threonine specific motifs that are substrates for either AMPK, Akt, PKA, PKC, or CDK kinases generally were higher than in the csCD150- samples. Since the mCD150 isoform with two ITSM signaling motifs in the cytoplasmic tail is predominant in CLL B cells, it may mediate signaling leading to the higher basal tyrosine and serine/threonine phosphorylation levels in csCD150+ CLLs. To assess this possibility, we stimulated csCD150+ CLLs with anti-CD150 mAb and compared levels of phosphorylation to unstimulated cells. CD150 ligation had no significant effect on the phosphorylation pattern either of AMPK, PKA, PKC or CDK substrates or any effect on global phospho-tyrosine levels. However, after CD150 ligation on csCD150+ CLL B cells, we detected an additional 40 kDa band in the phospho-Akt substrates suggesting that the Akt pathway was activated. Indeed, after ligation of CD150 on csCD150+ B cells Akt was phosphorylated at S473 and T308.
Since CD150 and CD180 showed the highest level of colocalization on the cell surface of CLL B cells, we assessed whether crosstalk between CD150 and CD180 mediated signaling pathways may happen. We performed simultaneous CD150 and CD180 coligation on CLL B cells in comparison with CD150 or CD180 crosslinking alone. CD150 or CD180 crosslinking on CLL B cells alone led to activation of Akt, mTORC1, ERK1/2, p38MAPK and JNK1/2 networks. Both CD150 and CD180 target the translation machinery through mTOR independent as well as mTOR dependent pathways. Moreover, both these receptors transmit pro-survival signals via Akt-mediated inhibition of GSK3β and FOXO1/FOXO3a. Unexpectedly, coligation CD150 and CD180 receptors on CLL B cells led to mutual inhibition of the Akt and MAPK pathways. While CD150 and CD180 coligation resulted in reduced phosphorylation of Akt, ERK1/2, c-Jun, RSK, p70S6K, S6RP, and 4E-BP; it led to
