Integrative Analysis of Ikaros-Dependent Changes of Transcriptional Regulation and Tyrosine Phosphorylation Events in Ph⁺ ALL

Tyrosine kinase-driven B-cell lineage acute lymphoblastic leukemia such as Ph⁺ ALL, is associated with particularly poor clinical outcome and defined by deletions of the IKZF1 gene encoding the Ikaros transcription factor in >80% of the cases. While the role of Ikaros in lymphoid differentiation has been extensively studied, it remains elusive how Ikaros functions as tumor suppressor in Ph⁺ALL. Here we demonstrate that Ikaros orchestrates a set of gene expression changes that affects leukemia cell proliferation and survival at multiple levels.

A comprehensive gene expression analysis revealed that reconstitution of Ikaros function in patient-derived Ph⁺ ALL cells mimics gene expression changes induced by Pax5 (a B cell specific transcription factor), μ-chain and BLNK (pre-B cell receptor components), and treatment with Imatinib (a BCR-ABL1 kinase inhibitor), while it reverses Myc- and Stat5a/b-dependent survival signals. In combining seven experimental perturbations (reconstitution of Ikzf1, Pax5, μ-chain, Blnk, treatment with Imatinib or deletion of Stat5a/b^fl/fl and Myc^fl/fl), we identified a commonly regulated gene set, including known tumor suppressors SPIB, BTG1, and BTG2 and highly enriched for Myc-related gene signatures. These findings suggest that reconstitution of PAX5 and IKZF1 transcription factors converges with pre-B cell receptor-mediated transcriptional changes. We next connected our analysis at the transcriptional level with Ikaros-dependent changes at the level of tyrosine phosphorylation events: The BCR-ABL1 kinase in Ph⁺ ALL drives proliferation and survival through phosphorylation of Stat5, AKT, ERK, SRC and multiple other signaling intermediates. A global mass spectrometry analysis of phosphotyrosine proteins in patient-derived Ph⁺ ALL cells showed that reconstitution of Ikaros leads to global loss of phospho-tyrosine, in a comparable manner as treatment with TKI. The vast majority of Ikaros-mediated changes resulted in loss of tyrosine phosphorylation such as the SRC tyrosine kinase LCK (Y504 and Y192), the proximal BCR-ABL1 adapter molecule NEDD9 (Y166, Y240, Y344 and Y578) and ABL1 itself (Y257). For a small number of molecules, Ikaros-reconstitution resulted in increased tyrosine phosphorylation including the phosphatases Shp2 (Ptpn11^Y584) and Ship2 (Inppl1^Y1161). A focus phospho-tyrosine analysis of individual signaling molecules analysis revealed that Stat5, SRC, AKT and ERK are all affected by Ikaros-induced de-phosphorylation. These results suggest that that Ikaros induces dephosphorylation of multiple signaling intermediates in BCR-ABL1 signaling. To identify Ikaros-mediated gene expression changes that were directly induced by Ikaros promoter binding, we matched Ikaros-dependent gene expression changes with Ikaros ChIP-seq data. This analysis confirmed enriched binding of Ikaros on Btg1, Myc and Smyd2 promoters. Among Ikaros target genes, we identified a set of genes that predicts clinical outcome for adult and pediatric patients with ALL. According to this predictor, Ikaros^UP (FBXO33, HBP1, HIVEP1, DTX1, KIF13B, RHOQ) and Ikaros^DOWN (PFKP, ASNS, ETV6, PSTPIP2, SMYD2, CDKL5), patients with B-ALL were segregated into two groups with significantly different outcomes, with a 5-year RFS of 76% vs 29% (COG P9906 trial, n=110; p=0.006) and a 5-year OS of 39% vs 0% (ECOG E2993 trial, n=25; p=0.007). Further biochemistry and mouse experiments showed that both the ability of Ikaros to dephosphorylate Stat5 and to function as tumor suppressor requires cooperation with the pre-B cell receptor linker molecule BLNK but not the proximal effector kinase Syk.

Importantly, Ikaros binds to and transcriptionally activates CDKN2A (Arf) and TP53 loci and thereby opposes the function of BCL6, which acts as transcriptional repressor at these loci. Comparing the gene expression microarray and ChIP-seq or ChIP-on-Chip data of BCL6 and Ikaros, we identified a set of target genes that is regulated by both BCL6 and Ikaros and found that for the vast majority of Ikaros/BCL6 target genes, Ikaros-reconstitution recapitulated a BCL6^−/− situation, i.e. genes that are induced by Ikaros are repressed by BCL6. These findings are of potential clinical relevance since pharmacological inhibition of BCL6 restores sensitivity of IKZF1-mutant Ph⁺ ALL cells to conventional tyrosine kinase inhibitors.

Druker:ARIAD: OHSU receives clinical trial funding. Dr. Druker is currently principal investigator or co-investigator on Novartis, Bristol-Myers Squibb, and ARIAD clinical trials. His institution has contracts with these companies to pay for patient costs, nurse and da Other; Bristol-Myers Squibb: OHSU receives clinical trial funding, OHSU receives clinical trial funding Other; Novartis: OHSU receives clinical trial funding, OHSU receives clinical trial funding Other; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Scientific Founder; OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. This potential individual and institutional conflict of interest has been reviewed and man, Scientific Founder; OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. This potential individual and institutional conflict of interest has been reviewed and man Other.