IKZF2, a Novel Target of MSI2 RNA-Binding Protein Plays an Oncogenic Role in Myeloid Leukemia

Deregulated epigenetic program is found in many cancers, and genetic aberrations of histone methyltransferases contribute to transformation in myeloid leukemias. Post-transcriptional regulation in leukemia has recently been highlighted as a novel way for maintaining the leukemia stem cell (LSC) program. We have recently demonstrated that Msi2 is required for LSC function in a murine MLL-AF9 leukemia model. We determined that MSI2 maintains the mixed-lineage leukemia (MLL) self-renewal program by interacting and retaining efficient translation of critical MLL regulated transcription factors including Hoxa9, Myc and Ikzf2. Despite extensive studies implicating Myc and Hoxa9 in leukemia, the role for Ikzf2 in myeloid leukemia is not known.

Ikzf2 is a member of the Ikaros transcription factor family and regulates lymphocyte development by controlling regulatory T-cell function. Ikzf2 is highly expressed in Hematopoietic Stem Cells (HSC) and to investigate if Ikzf2 is involved in HSC function, we utilized mice that have a specific deletion of Ikzf2 in the hematopoietic system through the Vav-cre system. We found similar frequencies for different populations in the stem, progenitor and mature cells in the bone marrow of Ikzf2^f/f and Ikzf2^D/D mice. Colony assays of isolated Lin^-Sca1⁺c-Kit⁺ (LSK) cells from Ikzf2^f/f and Ikzf2^D/D mice resulted in a comparable number of myeloid progenitor colonies. Furthermore, noncompetitive transplant of Ikzf2^f/f and Ikzf2^D/D bone marrow cells showed similar chimerism after 34 months indicating that Ikzf2^D/D mice have normal HSC function and hematopoiesis.

To interrogate the role of Ikzf2 in acute myeloid leukemia we utilized the MLL-AF9 retroviral transduction model.  Intracellular flow cytometry showed that IKZF2 is highly expressed in the LSC population compared to the non-LSCs.  We then transduced Ikzf2^f/f and Ikzf2^D/D LSK cells with MLL-AF9 and found that Ikzf2 deletion results in a ten-fold reduction in colony formation compared to Ikzf2^f/f cells.  Transplantation of transduced cells results in delayed leukemia progression with reduced disease burden. Secondary transplantation of the initiation experiment exhibited a significant delay in leukemogenesis in the Ikzf2^D/D compared to the Ikzf2^f/f mice (median survival of 32 and 19.5 days, respectively). The role for Ikzf2 in maintenance was assessed with an inducible puro-creER system, which resulted in 80% decrease in viable cell number within 24hrs of 4-hydroxytamoxifen (4-OHT) treatment. Flow cytometric analysis showed that the Ikzf2-deficient cells had increased apoptosis and differentiation, shown by AnnexinV/7-AAD and Mac1 expression respectively. Furthermore, inducible deletion of Ikzf2 using puro-creER system in vivo revealed that Ikzf2 deletion leads to a delay in leukemia after tamoxifen administration in mice. These results indicate that Ikzf2 is required for both leukemia initiation and maintenance.

To determine a role for IKZF2 in human leukemia cells, we performed Ikzf2 knockdown experiments with shRNAs in Kasumi-1, KG1, KCL22 and MOLM13 cells. Ikzf2 depletion resulted in decreased cell growth and increased apoptosis compared to cells infected with scramble shRNA.

To determine the mechanism for how IKZF2 controls leukemia cell survival and self-renewal, we performed gene expression profiling of the Ikzf2-deficient Vav-cre LSCs and demonstrated enrichment in signatures for self-renewal loss, increased differentiation, loss of Myc-regulated genes and loss for targets of Hoxa9 and Meis1. Further analysis overlapping our MSI2 HITS-CLIP data and our differentially regulated genes revealed a strong enrichment suggesting that the MSI2 bound targets are transcriptionally regulated by IKZF2. Lastly, ATAC-sequencing of Ikzf2^f/f and Ikzf2^D/D LSCs revealed alterations in chromatin accessibility that correlated closely with differentially expressed genes. Utilizing the ATAC-seq data we predicted that HOXA9 and MYC sites were significantly altered. We validated that MYC RNA and protein levels were reduced in both murine and human AML cell lines.

In contrast to its known tumor suppressor role in hypodiploid B-ALL and T-ALL, these results suggest that Ikzf2 contributes to MLL leukemia cell initiation and maintenance. Thus, we provide evidence that Ikzf2 can regulate c-MYC expression helping in maintaining the stem cell self-renewal program in LSCs.