SYK Is a Tumor Suppressor In Pre-B Cell Acute Lymphoblastic Leukemia and Not a Therapeutic Target
Authors: Ng Carina , Nahar Rahul , Elliott Emily , Lowell Clifford A. , Muschen Markus 
Published: October 2019

Abstract
Abstract 4199

Background:
The pre-B cell receptor promotes differentiation of normal pre-B cells and couples the immunoglobulin μ-chain to activating tyrosine kinases (e.g. SYK) via linker molecules (e.g. BLNK). We recently established that the pre-B cell receptor functions as a tumor suppressor in pre-B acute lymphoblastic leukemia (ALL) including ALL cells carrying the BCR-ABL1 oncogene (Trageser et al., J Exp Med, 2009). In virtually all cases of BCR-ABL1 ALL, pre-B cell receptor function is compromised and its reconstitution induces rapid cell cycle arrest. Given that the SYK tyrosine kinase represents a critical signaling molecule in the pre-B cell receptor pathway, one would expect that SYK tyrosine kinase activity has a tumor suppressive effect. It therefore seems counterintuitive that pharmacologic targeting of SYK was recently proposed as a new treatment approach for pre-B ALL (Uckun et al., Br J Haematol. 2010). While there is solid evidence for a role of Syk as a target in B cell lymphoma (Friedberg et al., Blood 2010) and B cell lineage CLL (Buchner et al., Blood 2010), where tonic B cell receptor signaling delivers critical survival signals, the role of Syk downstream of the pre-B cell receptor in ALL is unclear.

Results:
To clarify the role of SYK downstream of the pre-B cell receptor in pre-B ALL, we performed a genetic experiment to inducibly delete the Syk gene in pre-B ALL cells. To this end, pre-B cells from Syk-fl/fl mice were propagated in the presence of IL7 and then transformed with retroviral BCR-ABL1 or MLL-ENL oncogenes. After transformation, pre-B leukemia cells were transduced with 4-hydroxy-tamoxyfen (4-OHT)-inducible retroviral Cre-ERT2 or an ERT2 empty vector control. After puromycin-selection of Cre-ERT2 and ERT2 transduced leukemia cells, Cre-ERT2 or the ERT2 control were induced by addition of 4-OHT and deletion of Syk was studied at different time points. As assessed by Western blot and PCR, deletion of Syk was near complete after two days and undetectable after six days. We then studied changes in cell viability upon inducible deletion of Syk: Acute deletion of the Syk tyrosine kinase had no significant impact on the viability of pre-B ALL cells, even after prolonged cell culture over several weeks. We then reasoned that the effect of Syk-deletion may be subtle yet important, so we studied in BCR-ABL1-transformed Syk-fl/fl pre-B leukemia cells whether Syk-deletion sensitizes to Imatinib-treatment. Deletion of Syk was again confirmed by Western blot, yet the dose-response curves to Imatinib-treatment were superimposable for Syk-fl/fl and Syk-del/del pre-B leukemia cells. We conclude that SYK does not contribute important survival signals in our mouse model for pre-B ALL, nor does deletion of Syk sensitize BCR-ABL1-driven pre-B leukemia cells to Imatinib-treatment.
We next investigated the counter-hypothesis that Syk functions as a tumor suppressor downstream of the pre-B cell receptor. To test this possibility, we tested the effect of forced pre-B cell receptor expression in the presence or absence of Syk. Syk-fl/fl and Syk-del/del pre-B leukemia cells were transduced with CD8/μ-chain or a CD8 empty vector control. The μ-chain represents the central component of the pre-B cell receptor. Forced expression of the CD8 empty vector control had no effect regardless of whether Syk was deleted or not. When pre-B cell receptor signaling was reconstituted in Syk-fl/fl cells by expression of CD/μ-chain, viability of the leukemia decreased by >80%. By contrast, deletion of Syk greatly attenuated the tumor suppressive effect of CD8/μ-chain expression and less than 25% of the leukemia cells underwent cell cycle arrest and cell death.

Background:
Genetic deletion of Syk unequivocally demonstrates that Syk does not deliver critical survival signals downstream of the pre-B cell receptor in ALL. This is unlike B cell lymphoma, where tonic signaling from the B cell receptor promotes cell survival via Syk (Friedberg et al., 2010; Buchner et al., 2010). On the contrary, in pre-B ALL, the Syk kinase mediates pre-B cell receptor-induced cell cycle arrest. These findings are in direct conflict with a recent report on the therapeutic usefulness of pharmacological inhibition of Syk in pre-B ALL cell lines (Uckun et al., Br J Haematol.; 149: 508-17; 2010). The compound (C-61) used in this study may have unrecognized off-target effects, which might account for the discrepancies.

Disclosures:
No relevant conflicts of interest to declare.