Lack of CD44 is associated with reduced MCL1 levels and diminished BCR kinase response. (A) A representative immunoblot of 4 independent experiments screening a panel of indicated proteins from whole splenocyte lysates (1 leukemic mouse of age ≥ 12 months per lane) shows most prominently the reduced levels of MCL1 protein in the CD44 ko genotype vs CD44 wt leukemias. (B) Left: Densitometric values of relative MCL1/Actin protein expression are charted for a larger number of animals. For better comparability across different immunoblots, means in the CD44 wt cohort are set as 100%. Mean ± SEM values were 100.0 ± 11.9 for Eµ-TCL1:CD44wt vs 24.3 ± 6.3 for Eµ-TCL1:CD44ko (P < .0001). Right: qRT-PCR from splenocyte RNA of leukemic mice (age ≥ 12 months) with MCL1 mRNA normalized to peptidylprolyl isomerase A (cyclophilin A = PPIA). Means in the wt cohort were set as 100%. Mean ± SEM of at least 3 independent experiments per 11 animals per genotype show reduced MCL1 mRNA levels in Eµ-TCL1:CD44ko mice (51.6 ± 4.8 vs 100.0 ± 13.7 in Eµ-TCL1:CD44wt; P = .003). (C) CD44 deficiency leads to diminished BCR kinase response in murine CLL cells. Splenocytes of 1 representative leukemic mouse (age ≥ 12 months) for each genotype (n = 2) were stimulated by BCR crosslinking with in-solution anti–mouse IgM antigen-binding fragment. Lack of CD44 in Eµ-TCL1:CD44ko splenocytes is associated with a reduced amplitude and earlier termination of BCR-stimulated pSRC, pSYK, pERK1/2, and pAKT induction. This is associated with a much less robust MCL1 induction in BCR-activated CD44ko leukemic cells. (D) Densitometric evaluation of pERK1/2/ERK1/2 and pAKT/AKT protein from immunoblots on 3 individual splenic isolates summarizes the kinetics of the significantly reduced early BCR-induced phosphokinase response in CD44-deficient murine CLL cells. *P = .028 and P = .028 at 5 minutes and **P = .008 and P = .003 at 15 minutes for pERK1/2 and pAKT induction, respectively.