Two different "tales" of ATG7: Clinical relevance to myelodysplastic syndromes.

Somatic mutations in U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) are associated with various cancers including myelodysplastic syndrome (MDS). Mutant U2AF1 promotes malignant transformation by inhibiting autophagy, partly as a result of alterations in the 3' tail of ATG7. This results in altered mitochondrial function, increased reactive oxygen species production, and genomic instability.

Abbreviations

acute myeloid leukemia

autophagy related 7

cleavage factor Im

chronic myelomonocytic leukemia

cleavage and polyadenylation

interleukin 3

myelodysplastic syndrome

polyadenylation site index

small airway

splicing factor 3B subunit 1

Serine/Arginine-Rich Splicing Factor 2

The Cancer Genome Atlas

U2 Small Nuclear RNA Auxiliary Factor 1

untranslated region

Zinc Finger CCCH-Type, RNA Binding Motif And Serine/Arginine Rich 2

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem/progenitor cell disorders characterized by peripheral blood cytopenia, ineffective and dysplastic hematopoietic differentiation, and a high rate of progression to acute myeloid leukemia (AML). In the last few years the use of next-generation sequencing has resulted in an exponential increase in our understanding of the molecular genetic basis of MDS, with more than 50 genes reported to carry recurrent somatic mutations in MDS clones. Perhaps the most surprising finding was the discovery that up to 50% of patients with MDS carry somatic mutations in genes involved in splicing of premRNA such as Splicing Factor 3B subunit 1 (SF3B1), Serine/Arginine-Rich Splicing Factor 2 (SRSF2), Zinc Finger CCCH-Type, RNA Binding Motif and Serine/Arginine Rich 2 (ZRSR2), or U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1). The mechanism by which mutations in splicing factors result in a clonal disease such as MDS is not clear. It is also not known whether the mutations in splicing factor genes promote transformation. We addressed both of these questions in our recent study (Park et al. 2016) using primary cells from bone marrow of MDS patients and Ba/F3 and small airway (SA) cell systems. We studied the transformation ability of the most frequent mutation, U2AF1, which is present in 12% of MDS patients and is associated with poor prognosis, not only in MDS but also in several other hematologic cancers such as AML and chronic myelomonocytic leukemia (CMML).

To address whether mutations in splicing factor genes promote transformation, mutant (U2AF1S34F) and wild-type (U2AF1WT) U2AF1 were stably expressed in interleukin 3 (IL3)-dependent murine Ba/F3 cells. We found that the mutated gene transformed Ba/F3 cells to become IL3-independent and that these cells were subsequently able to form tumors in immunocompromised mice. Transcriptome profiling to study the functional consequence of these mutations revealed that cells expressing U2AF1S34F showed defective processing of many pre-mRNAs. Most interestingly, and unexpectedly, the defect was not altered splicing alone but also involved the differential selection of cleavage and polyadenylation (CP) sites in the 3′ untranslated region (UTR) of several genes, resulting in mRNAs with different 3′ tails. The CP selection of mRNA is determined by the cleavage factor Im (CFIm) protein complex via its 2 subunits CFIm59 and CFIm68, which respectively increase or repress the use of proximal CP sites. U2AF1S34F showed a decrease in binding to CFIm59 but not to CFIm68. This decreased binding with CFIm59 resulted in decreased use of the proximal site and increased use of the distal site, producing an elongated tail that ultimately caused translational repression of certain mRNAs. One such mRNA with an elongated tail and decreased protein levels due to translational repression corresponded to the ATG7 gene, which plays an essential role in autophagy. Autophagy, which literally means “self-eating”, is a cellular process that recycles intracellular proteins and organelles, thus avoiding a buildup of cellular waste products. Autophagy plays a very important role in cellular homeostasis, hence any defect in this process can result in a diseased state including cancer. Both an increase in autophagy and a decrease in autophagy can promote cancer, apparently in a context-dependent manner. In the case of transformation by mutant U2AF1, a decrease in autophagy due to a decrease in ATG7 protein promoted transformation. This transformation was accompanied by mitochondrial dysfunction, increased reactive oxygen species (ROS), and increased genomic instability as manifested by an increase in spontaneous mutational frequency.

The disease-related relevance of the above results was confirmed in clonal primary bone marrow cells from a large number of MDS patients with and without U2AF1S34F. An increased use of the ATG7 distal CP site compared to the proximal site was observed in MDS patients with U2AF1S34F. Furthermore, computational analysis of AML in The Cancer Genome Atlas (TCGA) data showed a significantly increased percentage of distal polyadenylation site index (PDUI) usage in mutants compared to the wild-type genotype. Previous studies have shown an increased production of ROS and mitochondrial damage, including an increase in mutations in mitochondrial DNA, in primary cells from MDS patients. Finally, mice that are deficient for Atg7 in the hematopoietic stem cell compartment develop severe anemia, lymphopenia, and other features resembling MDS/AML.

The therapeutic implications of these results come from observations that cells expressing U2AF1S34F are more sensitive to cytotoxic agents, including etoposide, 5-azacytidine, topotecan, and X-ray irradiation, suggesting that patients with U2AF1 mutations may respond better to cytotoxic agents. A prospective clinical trial of cytotoxic agents in patients with U2AF1 mutations may answer this question. However, the observation that deficient autophagy is required for initiation of transformation but not for maintenance of the disease implies that autophagy-targeting agents may not be particularly helpful.

The observations reported in this paper are significant for several reasons as the study (1) provides the molecular mechanism underlying the promotion and development of cancer by the most frequent splicing factor mutation p.S34F in the U2AF1 gene, (2) underscores the importance of deficient autophagic processes in promoting transformation, and (3) offers a novel mechanism involving extension of the 3′ tail of mRNA.