Abstract: Multiple sclerosis, a chronic autoimmune inflammatory disease associated with demyelination of the
central nervous system (CNS), remains a public health issue. Currently there is no known cure for multiple
sclerosis. Although several disease-modifying treatments (DMTs) are available, relapsing of multiple sclerosis
occurs frequently and DMTs often result in severe adverse effects such as liver failure and fetal outcomes. Novel
therapies are needed to reduce the disease burden for multiple sclerosis patients. Recently, we published that
Hectd3, an E3 ubiquitin ligase, is expressed predominantly in T cells of the immune system, which play a critical
role in pathogenicity of experimental autoimmune encephalomyelitis (EAE), a mouse model of human multiple
sclerosis. Specifically, we found that Hectd3 controls pathogenic Th17 effector response in EAE by regulating
ubiquitination of Malt1 and Stat3 in a non-degradative manner, resulting in stabilization of Malt1 and Stat3. In
addition, Hectd3-mediated polyubiquitination of Stat3 promotes Stat3 activation. Moreover, Hectd3-deficient
mice showed reduction in EAE disease scores, Th17 cell pathogenicity and effector Th17 cytokines.
Furthermore, Hectd3 deficiency causes a cell-intrinsic defect in Th17 cell pathogenicity that is responsible for
the attenuation of EAE in Hectd3-/- mice. Overall, our results demonstrate that Hectd3 is a critical modulator of
Malt1 and Stat3 signaling in EAE. Based on these results, we hypothesize that compounds abolishing Hectd3-
mediated ubiquitination of substrates can lower EAE severity. However, although Hectd3 plays significant roles
in pathogenesis of multiple sclerosis, currently there is no chemical probe to further investigate the pathways
and the implication in therapy of multiple sclerosis. Therefore, in this proposal, we aim to develop high throughput
screening assays to identify and characterize chemical probes to investigate in depth the biochemistry of Hectd3-
mediated Malt1 and Stat3 signaling pathways, and their therapeutic potential in pathogenic Th17 cells and EAE.
This innovative work explores the novel function of Hectd3 in immune regulation, specifically in pathogenic
Th17 cells, the identification of Malt1 and Stat3 as target substrates for Hectd3-mediated ubiquitination, and
characterization of novel chemical probes for Hectd3, and their impact on EAE. The long-term sustained
impact of this work is to identify compounds to modulate Hectd3 activity on its target substrates and its functions
in EAE to open avenues for development of more specific and effective immune therapies to treat multiple
sclerosis, a crucial need given current treatment challenges and limited therapeutic options. These combined
approaches will lead to the development of unique Hectd3 inhibitors with novel inhibition mechanisms. This work
will have a global reach by promoting fresh and effective strategies to treat multiple sclerosis. Hectd3 has also
been implicated in promoting breast cancer drug resistance, cancer metastasis (unpublished results), and
bacterial infections. Therefore, this project may also have significant impact on cancers and bacterial defense.