INPP4B is a Biomarker of Poor Prognosis in AML Which is Associated with EVI1 Overexpression and a LSC Signature

Acute myeloid leukemia (AML) is a highly heterogeneous cancer of the bone marrow. To better understand leukemogenesis and improve predictors of patient response to chemotherapy and overall survival (OS), we recently examined the role of inositol polyphosphate-4-phosphatase, type-II (INPP4B) in AML. Normally, INPP4B plays a role in PI3K/Akt signaling by regulating phosphorylation of phosphoinositides (PIs), critical membrane-bound second messenger molecules, by dephosphorylating the 4'-position of PI(3,4)P₂ to generate PI(3)P. Because PI(3,4)P₂, like PI(3,4,5)P₃, is necessary for the activation Akt, INPP4B was first hypothesized and demonstrated to be a tumor suppressor protein, akin to PTEN, in several cancers including breast, prostate and ovarian. Recent work however, including our own study, has demonstrated a paradoxical tumor-promoting role of INPP4B in AML and estrogen receptor positive (ER+) breast cancer. Specifically, we demonstrated that INPP4B^high AML patients (25% of patients) had significantly shorter OS, lower response to induction therapy, and shorter event-free survival. Furthermore, INPP4B expression was found to be an independent prognostic marker for OS in AML outperforming FLT3-ITD and NPM1 mutation status. Overexpression of INPP4B in several AML cell lines results in enhanced colony formation potential, chemotherapy drug resistance, and increased proliferation.

Though this previous work has shown that INPP4B plays a significant role in AML, it remains unclear why INPP4B^high AML differs from INPP4B^low AML, and what causes its upregulation. To address this question, we interrogated gene expression data from three independent datasets (n=942) to identify genes with differential expression between INPP4B^high and INPP4B^low AML. Gene expression analysis revealed that INPP4B^high AML was associated with differential expression of 233 genes. High INPP4B expression was associated with higher expression of genes related to the hematopoietic lineage, PI3K-Akt signaling, Jak-STAT signaling and ECM-receptor interaction pathways. Specifically, INPP4B^high AML has significantly higher expression of leukemic stem cell signature (LSC) genes CD34, RBPMS, GUCY1A3, KIAA0125, SOCS2, SPINK2, HTR1F, PPP1R16B, EVI1 (MECOM), DAPK1, BAALC, ABCB1, and PRKCH. Furthermore, INPP4B was found to be co-expressed with anti-apoptotic genes of the BCL2 family, namely BCL2 and BCL2L1. Further analysis revealed that INPP4B was co-expressed with the transcription factors EVI1, GATA2, NFATC2, ZEB1, GATA3 and ETS1, all of which having predicted binding sites within the INPP4B promoter region.

Due to the observed enrichment of hematopoietic lineage/LSC genes in INPP4B^high AML, we wanted to validate the potential role of the EVI1 transcription factor in regulating INPP4B expression. Chromatin immunoprecipitation was used to demonstrate that EVI1 binding was enriched in the INPP4B promoter region of both EVI1^high OCI/AML-4 and OCI/AML-6 cell lines. In addition, retroviral overexpression of EVI1 in EVI1^low U937 cells resulted in subsequent upregulation of INPP4B transcript levels. Moreover, shRNA mediated knockdown of EVI1 in EVI1^highOCI/AML-4 and UCSD-1 cells resulted in downregulation of INPP4B expression.

Overall, our analysis reveals that INPP4B^high AML is characterized by upregulation of genes related to the hemotopoietic lineage, and LSC signature - consistent with the in vitro colony formation phenotype seen in INPP4B overexpressing AML cell lines. Furthermore, we demonstrate that one of the hematopoietic stem cell genes, EVI1 is a potentially key regulator of INPP4B expression in AML.