Brian Liau, PhD, Harvard University Webinar

Presentation & Panel Discussions

Moderator: Brian Liau, Ph.D., Associate Professor of Chemistry and Chemical Biology, Harvard University

Presenters: Amanda Waterbury and Allyson Freedy

**Presenters’ Bios: **

Amanda Waterbury is a sixth-year PhD student in the Chemistry and Chemical Biology program in the Liau lab, where she is studying the mechanism of action of epigenetic inhibitors. Before pursuing her PhD studies, Amanda performed her undergraduate work with Prof. Richard Jordan at the University of Chicago, where she studied bimetallic catalysts scaffolds towards CO2 incorporation into ethylene polymers. While at the University of Chicago, she also investigated potential genes critical to the lethality of MRSA in the Duam Lab.

Ally Freedy is a MD/PhD student in the Harvard-MIT MD/PhD program and fifth-year graduate student in the Harvard Chemical Biology program working with Professor Brian Liau, where she has developed the method CRISPR-suppressor scanning to study resistance mutations to PRC2 inhibitors in EZH2-mutant lymphoma. Ally performed her undergraduate training with Professor Stuart Schreiber where she studied the mechanism of action of a Class IIA selective HDAC inhibitor. Subsequently she was awarded a Gates Cambridge scholarship to pursue MPhil studies at the University of Cambridge working with Professor Gonçalo Bernardes to develop a novel protein modification reaction.

**Abstract: **

Complexes of biological molecules participate in intricate interactions to regulate fundamental life processes. These interactions span different shapes, sizes, and length scales, from shape-complementary surfaces that comprise stable protein complexes to weak chemical forces that give rise to biomolecular condensates. These breadth and diversity are exemplified by chromatin complexes, the orchestrated interactions of which control gene expression and cell fate decisions. Dysregulation of these complexes and their interactions underlie human disease, and new therapeutic approaches to modulate them by disrupting or even creating new interactions are transforming paradigms for drug discovery. Unraveling the function and regulation of these complexes and their interactions requires integrating a deep understanding of their molecular architectures and dynamic features in cells.

To bridge these gaps, the Liau lab combines chemical biology with genomic technologies to illuminate protein complexes and their functions. By combining genome-editing with chemical inhibitor profiling in an approach called CRISPR-suppressor scanning, we describe the systematic identification of drug resistance alleles across protein targets, including chromatin complexes. These drug resistance alleles not only confirm on-target engagement but can be used as powerful discovery tools to uncover new principles and aspects of chromatin complex biology. We showcase these capabilities through two vignettes exploring (1) lysine-specific histone demethylase-1A (LSD1) and (2) the Polycomb Repressive Complex 2 (PRC2). Taken together, application of CRISPR-suppressor scanning in combination with detailed mechanistic investigations have led to unexpected biological discoveries that reshape approaches toward therapeutically targeting chromatin complexes.

Talk 1: Illuminating the functions of LSD1 complexes in leukemia using drug resistance alleles

Amanda Waterbury will discuss the group’s work towards clarifying the mechanism of LSD1 inhibitors in acute myeloid leukemia (AML). Using CRISPR-suppressor scanning, we identified several mutations in LSD1 that confer resistance to LSD1 active site inhibitors but unexpectedly impair LSD1 enzyme activity. We then demonstrated that LSD1 inhibitors exert their antiproliferative effects by disrupting a protein-protein interaction between LSD1 and GFI1B, a key hematopoietic transcription factor (TF), revising their mode of action. Furthermore, we identified several mutations in LSD1’s intrinsically disordered region (IDR) that confer drug resistance by impairing AML differentiation. Using a combination of genomic and microscopy approaches, we demonstrate that the IDR of LSD1 modulates LSD1-TF interactions, impacting enhancer activity and differentiation pathways in AML in response to LSD1 inhibitors. Through the characterization of these drug resistance mutations, we revise key roles and functions of LSD1 in maintaining enhancer landscapes and the AML cell state through its interactions with key hematopoietic TFs.

Talk 2: Drug addiction mutations unveil a methylation ceiling in EZH2-mutant lymphoma

Ally Freedy will present the lab’s work to characterize resistance mutations to clinical PRC2 inhibitors in lymphoma. Using CRISPR-suppressor scanning, we identify mutations in PRC2 that confer both resistance and drug addiction, resulting in lymphoma cells that paradoxically depended on the drug for survival. Drug addiction is mediated by hypermorphic mutations in the PRC2 methyltransferase EZH2 such that drug removal leads to overspreading of H3K27me3, a repressive epigenetic mark, surpassing a repressive methylation ceiling compatible with lymphoma cell survival. Through the characterization of these addiction mutations, we show that the activating EZH2 cancer mutations found in lymphoma establish an epigenetic state precariously close to this ceiling, which we show can be breached by inhibition of SETD2, a PRC2 antagonist, to block lymphoma growth. Through this work, we identify the first drug addiction mutation to an epigenetic therapy and in doing so identify a novel, targetable therapeutic vulnerability in EZH2-mutant lymphoma.