Investigating the Roles of ACLY in Multiple Myeloma Biology and Drug Sensitivity Open Access

Abstract

The gene ACLY codes for the cytosolic protein ATP citrate lyase (ACLY), the protein responsible for the conversion of citrate into acetyl-coenzyme A (acetyl CoA) and oxaloacetate (OAA). Acetyl CoA is used in the fatty acid synthesis pathway, the mevalonate pathway, and acetylation reactions. Many cancerous tumor cells deregulate cellular metabolism and display upregulation of ACLY and increased fatty acid synthesis, which is important for endogenous formation of saturated fatty acids. These fatty acids are used in the formation of cell membranes and membrane bound organelles, such as the endoplasmic reticulum (ER), a critical component of the secretory pathway. In multiple myeloma, the myeloma cells secrete copious amounts of monoclonal antibody, so we reasoned that the myeloma cells could be functionally dependent on ACLY. An analysis of the MMRF CoMMpass database showed that patients who expressed high quantities of ACLY mRNA had significantly worse progression free and overall survival compared to patients who expressed low quantities of ACLY mRNA. In the Broad Institute’s Cancer Dependency map, we found that myeloma cells are more dependent on ACLY than cell lines derived from other cancer types. To date, no studies have demonstrated the function of ACLY in multiple myeloma. The present study uses CRISPR-Cas9 single-guide RNA (sgRNA) lentiviral vectors to create ACLY knockout (KO) myeloma cell lines as well as the ACLY chemical inhibitor SB-204990 to investigate the impact of ACLY loss of function on myeloma cell proliferation, survivability, and response to treatment. of the greatest hindrances to multiple myeloma treatment is drug resistance, so it is critical to continue researching new ways to target myeloma’s plasma cell and cancer biology to improve standard of care.

Table of Contents

Table of Contents

Introduction......................................................................................................................................................................................................................................................... 1

Multiple Myeloma Epidemiology............................................................................................................................................................................................................................ 2

Clinical Characteristics of Multiple Myeloma........................................................................................................................................................................................................... 2

B-cell Development and Plasma Cell Differentiation................................................................................................................................................................................................ 4

Genetics of Multiple Myeloma................................................................................................................................................................................................................................ 5

Current Treatments for Multiple Myeloma.............................................................................................................................................................................................................. 8

Cancer Metabolism.............................................................................................................................................................................................................................................. 10

ATP Citrate Lyase................................................................................................................................................................................................................................................ 13

Scope of Thesis.................................................................................................................................................................................................................................................... 17

Materials and Methods......................................................................................................................................................................................................................................... 18

Cell Lines and Culture Conditions......................................................................................................................................................................................................................... 21

sgRNA Design..................................................................................................................................................................................................................................................... 21

Competent Cell Transformation and Plasmid Purification....................................................................................................................................................................................... 22

Transfection of HEK293T Cells............................................................................................................................................................................................................................. 22

Lentiviral Transduction........................................................................................................................................................................................................................................ 22

Western Blot Analysis.......................................................................................................................................................................................................................................... 23

Single Cell Cloning.............................................................................................................................................................................................................................................. 24

Quantitative Real-Time PCR................................................................................................................................................................................................................................. 24

Sequencing of Genomic DNA................................................................................................................................................................................................................................ 24

PicoProbe Acetyl-CoA Fluorometric Assay............................................................................................................................................................................................................. 25

Cell Death Assay.................................................................................................................................................................................................................................................. 25

Cell Proliferation Assay........................................................................................................................................................................................................................................ 26

Results................................................................................................................................................................................................................................................................ 27

Cancer Dependency Map analyses show most HMCLs exhibit dependency on ACLY.................................................................................................................................................. 28

Custom sgRNA sequences were cloned into pLX_sgRNA between the Xho1 and Nhe1 restriction sites....................................................................................................................... 33

ACLY was knocked out in OCI-My5 Cas9 cells after one week of Cas9 induction, but at two weeks ACLY expression increased..................................................................................... 34

ACLY was knocked out in OCI-My5 Cas9 and LP1 Cas9 cells after one week of Cas9 induction................................................................................................................................... 35

LP1 sgACLY2 and LP1 sgACLY5 cells were single cell cloned. One line appeared to have almost complete knockout, while three others expressed about half the protein of the control sample................................................................................................................................................................................................................................................................ 36

Quantitative real-time PCR on LP1 sgACLY2 and LP1 sgACLY5 single cell clones showed no decrease in mRNA expression in any of the single cell clones............................................ 37

Genomic DNA did not show any appreciable alterations in regions targeted by the sgRNA in any of the single cell clones........................................................................................... 37

LP1 cells treated with various concentrations of SB-204990 showed very little change in acetyl-CoA concentration.................................................................................................... 39

SB-204990 slowed cell growth and induced apoptosis in LP1, MM.1s, and OCI-My5 cells.......................................................................................................................................... 40

Combination treatment of SB-204990 and bortezomib induced more cell death in LP1 and KMS27, and at lower doses of bortezomib in MM.1s and OCI-My5....................................... 44

Discussion.......................................................................................................................................................................................................................................................... 46

References.......................................................................................................................................................................................................................................................... 55

Appendix I: Protocols and Recipes........................................................................................................................................................................................................................ 63

Appendix II: Primers and Plasmids....................................................................................................................................................................................................................... 98

About this Master's Thesis

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Cancer Biology
Degree	M.S.
Submission	Master's Thesis
Language	English
Research Field	Biology, Genetics Biology, General Biology, Cell
Keyword	ACLY Multiple Myeloma
Committee Chair / Thesis Advisor	Lawrence Boise, Emory University
Committee Members	Malathy Shanmugam, Emory University Gregory Lesinski, Emory University

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