PROJECT SUMMARY

Myelodysplastic syndrome (MDS) is a diverse malignant disease of the bone marrow as a result of defective blood-forming cell, otherwise known as hematopoietic stem cells (HSC). MDS is most prominent in individuals over 60 years of age, and as a result of longer life expectancies, the incidence of MDS has escalated in recent years. MDS progresses to Acute Myeloid Leukemia (AML) or bone marrow failure (BMF); therefore, the most effective therapies will likely target and eliminate the defective HSC, thus restoring normal blood formation. For many genes implicated in MDS, it is not known how they contribute to HSC dysfunction, abnormal blood cell production, and development to AML, and this gap in knowledge impedes the development of target-specific therapeutics. Current treatment options for MDS patients remain limited with no effective cure; therefore, there is an urgent need for new treatments. Furthermore, the complexity and heterogeneity of MDS, and the lack of mouse models, remain as obstacles to understanding and effectively treating this disease.

Recent progress in genomic technologies has led to many discoveries and a better understanding of the disease mechanisms in MDS. In addition, population-based analyses revealed that chronic immune stimulation increases the risk for MDS. The relevance of the immune pathway to MDS is further supported by studies showing that innate immune pathway genes are abnormally expressed in MDS HSC cells. Moreover, chronic TLR activation impairs hematopoiesis and contributes to premalignant conditions, including MDS, for which the cellular and molecular basis is unknown. Multiple independent genetic mechanisms contribute to TLR activation in MDS hematopoietic cells, which converge on the central mediator, TRAF6. TRAF6 is a downstream TLR-effector ubiquitin (Ub) ligase, which is overexpressed, and implicated in the pathogenesis of MDS and related myeloid leukemias.

The project supported by the Evans Foundation revealed a novel mechanism involving directed ubiquitination of an RNA binding protein and auxiliary spliceosome factor (hnRNPA1) by TRAF6 and the consequences of hnRNPA1 ubiquitination on RNA processing of selective genes. We established that chronic TRAF6 signaling, as described in human MDS or after LPS stimulation, results in aberrant RNA splicing of hematopoietic-requisite genes, and hematopoietic defects consistent with human MDS. Specifically, we describe that aberrant splicing of Arhgap1, a GTPase activating protein, leads to constitutive Cdc42 GTPase activation and causally to hematopoietic dysfunction in TRAF6-expressing cells. Mechanistically, our data show that TRAF6 directly ubiquitinates hnRNPA1, which affects hnRNPA1 binding to RNA and exon skipping. A summary of the key implications is described below:

1. Cell-intrinsic TRAF6 activation, either by TLR stimulation or overexpression as in MDS, regulates hematopoietic stem/progenitor function as a result of aberrant RNA exon usage.
2. Through a global Ub-enrichment screen to uncover novel TRAF6 substrates, we identified and interrogated an auxiliary spliceosome factor, mechanistically linking TRAF6 to RNA processing.
3. During chronic TRAF6 signaling, missplicing of Arhgap1 results in Cdc42 activation and several of the hematopoietic defects associated with MDS, which can be reversed by inhibiting Cdc42.
4. hnRNPA1 ubiquitination by TRAF6 adjacent to its RNA-binding domain regulates the affinity of hnRNPA1 for Arhgap1 pre-mRNA, Arhgap1 exon 2 retention, and also hematopoietic function.

As previously reported, hematopoietic and immune cells orchestrate exon/intron usage during normal differentiation (Cell, 2012) or following TLR stimulation (Nature, 2012); however, the underlying mechanism up until now is not known. As such, our findings make fundamental insights by uncovering a mechanism linking immune signaling and control of RNA processing through ubiquitin modification of RNA processing factors by TRAF6 in human and mouse cells.

In summary, we believe our findings have transformative implications in understanding how normal and chronic TLR signaling affects hematopoietic cell function through regulation of RNA processing (i.e., splicing) while also contributing to premalignant conditions, such as MDS. Our findings are also significant because they connects two common genetic alterations in MDS: chronic immune pathway activation and altered mRNA splicing. We anticipate that the knowledge gained from these studies will advance our understanding about the molecular and cellular basis of MDS, and uncover novel treatment strategies.