Molecular Pathogenesis of Progressive Refractoriness in MLL -Rearranged AML

Background

Despite advanced therapeutics, many leukemia patients become refractory to additional therapy, accounting for a major cause of leukemic deaths. Among major human acute myeloid leukemias (AMLs), MLL -rearranged AML is characteristic of progressive refractoriness to chemotherapy and shorter period to relapse. To clarify the molecular pathogenesis of such characteristics, we analyzed genetic abnormalities accumulated in MLL -rearranged AML in additional to the genetic events like MLL -fusion and MLL -partial tandem duplication (MLL-PTD).

Method

We exploited a mouse model of recurrent leukemia generated by mouse progenitors transduced with human MLL / AF9. Mouse leukemic cells with MLL / AF9 were serially transplanted in a total of 58 mice in 4 generations. We investigated somatic genetic events in leukemic cells from mouse models of MLL -rearranged AML and patients with MLL -rearranged AML. Whole exome and targeted-capture sequencing results were combined and analyzed to detect somatic genetic events and whole transcriptome sequencing and microarray dataset to assess gene expression.

Results

In two independent experiments using mouse with transduced MLL/AF9, leukemic cells obtained in the 1st (N=2) and 4th (N=9) transplants were analyzed by whole exome sequencing. The onset of leukemia became progressively earlier in later generation transplant. Median periods to relapse were 57 and 16 days in the 1st and the 4th transplants, respectively (P < 0.001). With regard to molecular pathogenesis, leukemic cells in the 4th transplants had 12−31 more mutations, in addition to the mutations found in those in the 1st transplants (6-44 mutations). Significantly, two RNA polymerases (Polr2a and Polr3d), which are functionally related to Mll, were mutated in two experiments during the 4th transplants, suggesting that genetic lesions in MLL-associated complex could play a synergetic role in survival advantage of MLL/AF9 positive cells.In addition, mutations in Ptpn11 (G60R) and Gnb2 (G77R), which correspond to the same residues in human proteins, were also detected. In the cohort of human hematological neoplasms analyzed by targeted-capture sequencing, PTPN11 (G60R) is located at the very well documented hotspot in MLL -rearranged human AML and GNB2 G77 residue is present close to that affected by other recurrent mutations in human AML. Of note is that a germline mutation of the identical position of orthologous protein GNB1 was most recently reported in a case with acute lymphoblastic leukemia. To confirm pathological significance of such a novel GNB2 mutation, we analyzed gene expression in human AML. When compared between human MLL -rearranged (N=176) and MLL -rearrangement (−) (N=692) AML cases, GNB2 transcripts were significantly upregulated in human MLL -rearranged AML cases. Functionally, we induced wild-type and G77R mutant GNB2 in human hematopoietic cell lines. Overexpression of wild-type GNB2 conferred cytokine independency to granulocyte macrophage colony-stimulating factor dependent leukemic cell line TF1. Induced GNB2 (G77R)and knocked down wild-type GNB2 in MLL -rearranged AML cell lines were used to assess and confirm the molecular pathogenesis associated with a novel candidate protein GNB2.

Conclusions

Our results in serial transplant experiments provide insights into the molecular mechanism of progression and progressive refractoriness of human MLL -rearranged AML. This study confirms that GNB2 is a novel candidate protein whose overexpression and activated mutation might play a significant role in the pathogenesis of this leukemia.