Illuminating the nonenzymatic functions of LSD1 complexes in leukemia using drug resistance alleles

Abstract

The identification of drug resistance conferring mutations not only confirms on-target small molecule mechanism but can also serve as a useful discovery tool to uncover novel aspects of target biology. However, the identification of drug resistance conferring mutations is difficult, especially in allosteric or scaffolding sites and with protein targets lacking structural data. Using a CRISPR-Cas9 tiling mutagenesis approach termed CRISPR-suppressor scanning to profile resistance mutations, we elucidated the downstream mechanism of lysine-specific histone demethylase 1 (LSD1) inhibitors in acute myeloid leukemia (AML). We demonstrate that this approach can rapidly identify mutations that confer drug resistance. Moreover, our studies illustrate how CRISPR-suppressor scanning can identify functional hotspots beyond the small molecule binding site that clarify the mechanism of small molecule inhibitors and allosteric sites essential for target protein function. In this thesis, I present three stories on the histone lysine demethylase LSD1 that illustrate the application of CRISPR-suppressor scanning as a tool for biological discovery. The first chapter introduces key concepts and examples, from chromatin biology to the identification of drug resistance conferring mutations. Recent advances in structurally resolving LSD1 and its complex members are reviewed as well as efforts to pharmacologically target LSD1 for oncology and neurological disorders. The following chapter discusses our work investigating resistance mutations generated by CRISPR-suppressor scanning to small molecule inhibitors of LSD1 in the context of AML. LSD1 was discovered as a vulnerability in cancer, where knockdown of LSD1 was shown to suppress leukemia cell growth, prompting interest in developing pharmacological inhibitors of LSD1. However, how LSD1 inhibition leads to leukemia cell growth arrest was unclear at the offset of our studies. By profiling resistance mutations generated by CRISPR-suppressor scanning to LSD1 small molecule inhibitors in AML cell lines, we demonstrate that the demethylase activity of LSD1 is not essential for cancer cell proliferation and that the antiproliferative activity of LSD1 inhibitors stems from disrupting the protein-protein interaction between LSD1 and the hematopoietic transcription factor, GFI1B. Overall, this study revised the mechanism of action of LSD1 small molecule inhibitors and clarified the scaffolding functions of LSD1 in leukemia. In chapter three, I describe our efforts towards elucidating the role of the disordered LSD1 N-terminus in modulating LSD1 function in AML. In the previous study, we identified mutations in the N-terminus of LSD1 that were enriched in the presence of LSD1 inhibitors. How mutations in this intrinsically disordered region (IDR) of LSD1 confer drug resistance was unclear, especially given that the LSD1 IDR is distal from the drug binding site and is not structurally resolved. Through mechanistic investigations of these drug resistant mutants, we identify LSD1’s role in the downstream mechanism of LSD1 inhibition, beyond LSD1-GFI1B complex dissociation. We found that LSD1 inhibitors are transcription factor reprogrammers, prompting the dissociation of the high-affinity LSD1-GFI1B complex to promote the formation of weaker protein-protein interactions with myeloid transcription factors to buffer enhancer activity. The LSD1 IDR mutants display increased interactions with key myeloid transcription factors, which we suggest prevents enhancer commissioning after LSD1 inhibitor treatment in these drug resistant cell lines. We further clarify the function of the LSD1 IDR in modulating transcription factor interactions and enhancer activity, revealing new aspects of LSD1 biology in the context of AML. In chapter four, I describe how a distal loop deletion in LSD1 mediates drug resistance by promoting the fragmentation of a covalent drug adduct, illuminating new mechanisms of drug resistance beyond the canonical perturbation of the drug binding site. The covalent drug fragmentation induced by this resistance mutation is mechanistically similar to demethylase specific LSD1 inhibitors, such as T-448, which are being tested for neuroscience applications. Through the study of this loop deletion mutant and T-448 analogs, we elucidate the mechanism and structural features that guide this covalent drug adduct rearrangement. Furthermore, this study highlights how mutations in allosteric or distal sites can promote resistance by unique mechanisms and will hopefully be informative for the development of therapeutics. Collectively, these projects showcase the utility of CRISPR-suppressor scanning in identifying drug-resistance conferring mutants, the mechanistic study of which can illuminate fundamental aspects of protein function, downstream biology, as well as small molecule mechanism of action. These findings not only advance our understanding of LSD1 in the context of AML but also provide fundamental insights into the role of chromatin modifiers beyond their canonical enzymatic functions and illustrate how both scaffolding and disordered sites can modulate their activity.