LCK over-expression drives STAT5 oncogenic signaling in PAX5 translocated BCP-ALL patients

Abstract

The PAX5 gene is altered in 30% of BCP-ALL patients and PAX5 chromosomal translocations account for 2-3% of cases. Although PAX5 fusion genes significantly affect the transcription of PAX5 target genes, their role in sustaining leukemia cell survival is poorly understood. In an in vitro model of PAX5/ETV6 leukemia, we demonstrated that Lck hyper-activation, and down-regulation of its negative regulator Csk, lead to STAT5 hyper-activation and consequently to the up-regulation of the downstream effectors, cMyc and Ccnd2. More important, cells from PAX5 translocated patients show LCK up-regulation and over-activation, as well as STAT5 hyper-phosphorylation, compared to PAX5 wt and PAX5 deleted cases. As in BCR/ABL1 positive ALL, the hyper-activation of STAT5 pathway can represent a survival signal in PAX5 translocated cells, alternative to the pre-BCR, which is down-regulated. The LCK inhibitor BIBF1120 selectively reverts this phenomenon both in the murine model and in leukemic primary cells. LCK inhibitor could therefore represent a suitable candidate drug to target this subgroup of ALL patients.

Figure 1. PAX5/ETV6 fusion protein leads to Lck over-activation

Lck is up-regulated in PAX5/ETV6 transduced LY5.1FL pre-BI cells both (A) in basal conditions (FC = 2.04) and (B) in time course experiments in synchronized cells after overnight withdrawal of IL7 (FC = 7.96, FC = 15.63, FC = 3.83, FC = 4.32, at 0 h, 24 h, 48 h and 72 h, respectively). (C) Lck protein expression and (D) schematic representation of LckY505/Lck ratio, summarizing n = 5 western blot experiments; paired t test, p < 0.05. (E) Csk and (F) Zap70 mRNA expression levels in LY5.1FL cells FC = 0.74 and FC = 6.8, respectively; test t, **p < 0.01; ***p < 0.001.

Figure 2. LCK is over-expressed in PAX5 translocated BCP-ALL cases

(A) PAX5 fusion genes (dark grey) induce LCK over-expression in primary patients samples, compared to PAX5 wt (white) and PAX5 deleted BCP-ALL patients (light grey) (PAX5 translocated cases FC = 6.27, test t, p < 0.05, PAX5 deleted cases FC = 3.23, n.s.). LCK over-expression is maintained in PAX5 translocated patient-derived xenograft BM cells (FC = 21.58, test t, p < 0.01). (B) Western blot analysis of LCK expression levels in PAX5 wt, PAX5 deleted primary patients cells and in PAX5 translocated patient-derived xenograft BM cells. (C) Schematic representation of LCK expression (mean = 0.08, 0.18, 0.17 (arbitrary unit) in PAX5 wt, PAX5 deleted, and PAX5 translocated cases, respectively) and (D) LCK^Y505/LCK ratio, (mean = 12.03, 9.15, 4.09 in PAX5 wt, PAX5 deleted, and PAX5 translocated cases, respectively). FC, fold change; xeno, PAX5 translocated patient-derived xenografts.

Figure 3. PAX5/ETV6 induces STAT5 hyper-activation in murine LY5.1FL pre-BI cells

(A) Representative dot plots of STAT5^Y694 FACS analysis. (B) RQ-PCR of cMyc and (C) Ccnd2 at early and late time points after IL7 administration (cMyc FC = 0.33, FC = 2.64 and FC = 3.78; Ccnd2 FC = 1.00, FC = 2.26 and FC = 1.83 at 0 h, 30 min and 24 h after IL7 administration, respectively). Test t, **p < 0.01; ***p < 0.001.

Figure 4. The Lck inhibitor BIBF1120 reverts the advantage of PAX5/ETV6

(A) Representative dot plots of STAT5Y694 FACS staining after administration of BIBF1120 in LY5.1FL cells and (B) relative schematic representation of the percentage of STAT5Y694 positive cells. (C) Increased number of replication cycles in PAX5/ETV6 transduced pre-BI cells is abrogated by BIBF1120 administration.

Figure 5. The Lck inhibitor BIBF1120 down-tunes STAT5 activation in PAX5 translocated cells

(A) Percentage of STAT5 activation in frozen blast cells at basal level and after stimulation with hIL7 (50 ng/ml for 30 minutes). Despite the low basal level with no biological differences in the patients' groups, stimulation with hIL7 induces significantly higher STAT5 activation in PAX5 translocated cells. (B) Overnight in vitro co-culture of blast cells on OP9 stroma confirmed that PAX5 translocated cells show a higher STAT5 activation profile compared to PAX5 wt and PAX5 deleted patients. Stimulation with hIL7 increased the level of STAT5^Y694 positive cells and PAX5 translocated cells maintained the highest activated profile. Overnight treatment with BIBF1120 is able to reduce STAT5 activation in PAX5 fusion gene positive cells, but it doesn't exert any significant effect on PAX5 wt and PAX5 deleted cells. Treatment with BIBF1120 abolishes the effect of stimulation with hIL7 in PAX5 translocated cases, keeping the percentage of STAT5 activated cells similar to the levels of the other groups. (C) LCK levels are inversely correlated to leukemia cells viability. Test t, *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6. Mechanism of action of PAX5 fusion proteins in BCP-ALL

The pre-BCR is not expressed in PAX5 translocated blast cells (A), causing the typical proliferation advantage and differentiation block of BCP-ALL (B). In particular, PAX5 fusion proteins cause the up-regulation of LCK and its over-activation (C). LCK over-activation leads to STAT5 hyper-phosphorylation (D), thus sustaining proliferation and survival of blast cells via the transcription of STAT5 target genes (E). The administration of the LCK inhibitor BIBF1120 reverts this phenomenon, down-tuning STAT5 signaling (F).