Identification of the Adapter Molecule MTSS1 as a Potential Oncogene-Specific Tumor Suppressor in Acute Myeloid Leukemia

Abstract

The adapter protein metastasis suppressor 1 (MTSS1) is implicated as a tumor suppressor or tumor promoter, depending on the type of solid cancer. Here, we identified Mtss1 expression to be increased in AML subsets with favorable outcome, while suppressed in high risk AML patients. High expression of MTSS1 predicted better clinical outcome of patients with normal-karyotype AML. Mechanistically, MTSS1 expression was negatively regulated by FLT3-ITD signaling but enhanced by the AML1-ETO fusion protein. DNMT3B, a negative regulator of MTSS1, showed strong binding to the MTSS1 promoter in PML-RARA positive but not AML1-ETO positive cells, suggesting that AML1-ETO leads to derepression of MTSS1. Pharmacological treatment of AML cell lines carrying the FLT3-ITD mutation with the specific FLT3 inhibitor PKC-412 caused upregulation of MTSS1. Moreover, treatment of acute promyelocytic cells (APL) with all-trans retinoic acid (ATRA) increased MTSS1 mRNA levels. Taken together, our findings suggest that MTSS1 might have a context-dependent function and could act as a tumor suppressor, which is pharmacologically targetable in AML patients.

Fig 1. MTSS1 is upregulated in t(8;21) and inv(16) AML patients.

The gene expression profiling of MTSS1, DNMT3B and DNMT3A in different AML subsets (GEO accession number GSE1159) is shown (A and B). MTSS1 mRNA levels were measured by qRT-PCR in human AML cell lines carrying either a FLT3-ITD mutation (MV4-11), t(15;17) (NB4), t(8;21) (Kasumi-1) or undefined genetic changes (U937) in relative expression to GAPDH (n = 3; C) which was verified at the protein level by Western Blot analysis using GAP-DH as a loading control (D).

Fig 2. MTSS1 expression correlates with better clinical outcome of normal karyotype-AML patients.

MTSS1 mRNA levels were measured in patient-derived AML samples (n = 66) with either a Normal Karyotype (NK, n = 38), t(15;17) (n = 15), t(8;21) (n = 5) or inv(16) (n = 8) aberration by qRT-PCR and are expressed as % of GAPDH (A). Using the Leukemia Gene Atlas [37] and data from the German AMLCG 1999 clinical trial [19], two groups of patients (above vs. below the median expression of MTSS1) were analyzed for overall survival (OS) in NK AML patients (n = 163, logrank test P = 0.03; GEO accession number GSE12417; B). Similarly, in the same trial, patients with a DNMT3B expression above vs. below the median were analyzed for survival (n = 163, logrank test P = 0.04; C). We then segregated patients into two groups based on their MTSS1 and DNMT3B expression levels according to high MTSS1 and low DNMT3B or low MTSS1 and high DNMT3B expression and assessed overall survival (AMLCG 1999, n = 163, logrank test P = 0.006; D). Binding of DNMT3B to the MTSS1 promoter was analyzed by chromatin immunoprecipitation experiments using NB4 and Kasumi-1 cells. Precipitated ChIP-DNA was quantified using real-time PCR and SYBR Green for MTSS1 promoter region -864/-645 (E).

Fig 3. MTSS1 expression is increased by ATRA treatment in t(15;17) AML.

MTSS1 mRNA levels were measured in human AML cells (U937) transduced with either empty vector control (PMT Control) or an inducible PML-RARα (PR9) after 24 hours of activation with zinc (Zn) by qRT-PCR (n = 3; A). Similarly, MTSS1 mRNA levels were measured after treatment with all-trans retinoic acid (0.5μM ATRA) or 0.5μM DMSO control after 48 and 72 hours in human AML cells carrying the PML-RARα translocation (NB4) (n = 3; B).

Fig 4. MTSS1 is positively regulated by AML1-ETO in human AML cells.

The gene expression of MTSS1 in human AML cells (Kasumi-1 without transduction (no Trans), scramble siRNA (ctr RNAi) or siRNA targeting the AML1-ETO translocation is shown (GEO accession numbers GSE15646; A). Similarly, the gene expression of MTSS1, DNMT3B and 3A are measured in a gene expression analysis of human cord-blood derived CD34⁺ cells and patient-derived AML samples with the AML-1 ETO translocation (GEO accession numbers GSE8023; B and C). Human AML cell lines carrying either a FLT3-ITD mutation (MV4-11), t(15;17) (NB4), t(8;21) (Kasumi-1) or not defined (U937) were transduced with a MTSS1 firefly / renilla construct and the MTSS1 promoter activity as a normalized relative ratio was measured (n = 3;D).

Fig 5. FLT3-ITD mediated suppression of MTSS1 is pharmacologically reverted by PKC-412.

Mtss1 mRNA levels were measured in murine 32D cells transduced with either empty vector control (EV) or a FLT3-ITD overexpression vector by qRT-PCR (FLT3-ITD; n = 3; A). Similarly, FLT3-ITD transduced murine 32D cells and human FLT3-ITD⁺ AML cells (MV4;11) were treated with the pharmacological FLT3-ITD inhibitor PKC-412 and DMSO control and MTSS1 mRNA levels were measured after 24 hours (n = 3; B and C).