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MYC is a key oncogenic driver frequently overexpressed in non-small cell lung carcinoma (NSCLC) and other cancers, where its protein levels often exceed what would be expected from MYC mRNA levels alone, suggesting post-transcriptional regulation. Strategies to inhibit MYC function by targeting mRNA translation hold potential for therapeutics utility in Myc-dependent cancers. We developed TranslationLight, a high-content imaging platform which detects MYC mRNA translation in human cells. Using this system, we conducted a high-throughput screen of ~100,000 compounds to identify small molecules that selectively modulate MYC translation. Candidate compounds were evaluated by immunofluorescence, ribosome profiling, RNA sequencing, cellular thermal shift assays (CETSA), and subcellular localization studies of mRNA and RNA-binding proteins. We identified a lead compound, CMP76, that potently reduces Myc protein without substantially decreasing its mRNA abundance. Mechanistic investigations showed that the compound induces relocalization of MYC mRNA into stress granules, accompanied by translational silencing. CETSA identified hnRNPK as a primary protein target, and compound treatment triggered its cytoplasmic relocalization together with formation of hnRNPK-containing granules colocalizing with MYC mRNA. Analysis across cancer cell lines revealed that sensitivity to CMP76 was significantly associated with RBM42 dependency. This work establishes a novel therapeutic strategy to inhibit MYC translation mediated by hnRNPK, offering a translationally targeted approach to cancer therapy.

Antibody engineering technology is at the forefront of therapeutic antibody development. The primary goal for engineering a therapeutic antibody is the generation of an antibody with a desired specificity, affinity, function, and developability profile. Mature antibodies are considered antigen specific, which may preclude their use as a starting point for antibody engineering. Here, we explore the plasticity of mature antibodies by engineering novel specificity and function to a pre-selected antibody template. Using a small, focused library, we engineered AAL160, an anti-IL-1β antibody, to bind the unrelated antigen IL-17A, with the introduction of seven mutations. The final redesigned antibody, 11.003, retains favorable biophysical properties, binds IL-17A with sub-nanomolar affinity, inhibits IL-17A binding to its cognate receptor and is functional in a cell-based assay. The epitope of the engineered antibody can be computationally predicted based on the sequence of the template antibody, as is confirmed by the crystal structure of the 11.003/IL-17A complex. The structures of the 11.003/IL-17A and the AAL160/IL-1β complexes highlight the contribution of germline residues to the paratopes of both the template and re-designed antibody. This case study suggests that the inherent plasticity of antibodies allows for re-engineering of mature antibodies to new targets, while maintaining desirable developability profiles. A proof of principle approach redirects an anti-IL-1b antibody to bind the otherwise unrelated antigen, IL-17A, highlighting the plasticity of antibody scaffolds that could be manipulated for alternative binding or function.

Protein degradation controls many fundamental processes, including cell cycle and signaling, DNA transcription and translation and is crucial for normal development and physiology [1]. The ubiquitin-proteasome system (UPS) and the autophagy pathway are the two major pathways responsible for the degradation and clearance of damaged or excess proteins and organelles in the cell [2]. Although the two pathways significantly differ in the molecular machinery they employ, ubiquitin appears to serve as a common signal in the two systems. In the first part of my thesis I studied the contribution of autophagy to protein degradation process under proteasomal overload. We show that antibody secreting B cells (D2 cells) have higher levels of ubiquitinated substrates compared to control pre-B cells (70z cells). Furthermore, inhibition of lysosomal degradation using BafilomycinA1 (BafA) in D2 cells led to accumulation of ubiquitinated substrates in a similar manner as inhibition of the proteasomal function using velcade. Thus demonstrating the existence of proteasomal overload under physiological conditions. To study proteasomal overload we knocked down two proteasomal ubiquitin receptors S5a and ADRM1 in HeLa cells, knockdown of S5a and ADRM1 led to reduction in the UPS flux, concomitantly with upregulation of p62-dependent autophagic degradation of ubiquitinated substrates. The short-lived transcription factor ATF4 accumulated under these conditions and led to upregulation in p62 mRNA level. These findings provide a molecular mechanism by which selective autophagy is upregulated in response to disruption in the normal flow of substrates to proteasomal degradation. These results clearly present new evidence for the cross talk between the UPS and the autophagic pathway. Interestingly, knockdown of S5a and ADRM1 reduced the accumulation of insoluble polyubiquitinated protein aggregates following proteasomal inhibition. In attempt to better characterize the relationship between the two proteolytic systems I describe in this part, a novel interaction between the HECT family E3 ubiquitin ligase NEDD4 and the autophagic ATG8 family members. I show that the WW region of NEDD4 and the N-terminal α helix of the ATG8s mediate a direct interaction between NEDD4 and mammalian ATG8s. Furthermore, NEDD4 coimmunoprecipitated with p62 in a Ca2+dependent manner. Knockout of Nedd4 increased p62 protein level and attenuated autophagic flux of LC3. Taken together this study sheds new light on the role of autophagy as a complementary system that can sense proteasomal function and balance it in a case of need, in addition we show that the UPS component E3 ubiquitin ligase NEDD4 has a regulatory role on the autophagic process.

HSAN9 is a rare progressive neurodegenerative disease in children linked to bi-allelic loss-of-function mutations in the TECPR2 gene. TECPR2 is a multi-domain protein harboring N-terminal WD repeats and C-terminal TECPR repeats, followed by a functional LIR motif that serves in autolysosomal targeting. Here, we show that the lack of TECPR2 leads to impairment of mitophagy that can be recovered by the expression of its C-terminal domain. Accordingly, we uncover severe mitochondrial dysfunction and accumulation of mitochondrial content in primary fibroblasts derived from an HSAN9 patient, and in embryonic fibroblasts and dorsal root ganglia derived from an HSAN9 mouse model. Strikingly, these mitochondrial defects are mediated by a mitochondrial stress through activation of the integrated stress response (ISR), whereas mitochondrial function is recovered by pharmaceutical or genetic suppression of ISR. Our findings provide a new link between mitophagy and ISR in mitochondrial homeostasis during neurodegeneration.