THE UNIVERSITY OF CHICAGO THE ROLE OF H3K79ME2 IN LEUKEMIA AND ALTERNATIVE SPLICING A DISSERTATION SUBMITTED TO THE FACULTY OF THE DIVISION OF THE BIOLOGICAL SCIENCES AND THE PRITZKER SCHOOL OF MEDICINE IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN CELL AND MOLECULAR BIOLOGY BY WILLIAM FRANK RICHTER, JR. CHICAGO, ILLINOIS MARCH 2021 Dedicated to my mother, who always supported me in my endeavors and helped spark my interest in science. TABLE OF CONTENTS LIST OF FIGURES ..................................................................................................................... viii LIST OF TABLES .......................................................................................................................... x ACKNOWLEDGEMENTS ........................................................................................................... xi ABSTRACT .................................................................................................................................. xii 1. INTRODUCTION .................................................................................................................. 1 HISTONE POST-TRANSLATIONAL MODIFICATIONS ......................................... 1 DOT1L AND METHYLATION OF LYSINE 79 OF HISTONE H3 ............................ 6 MLL-REARRANGEMENTS AND HISTONE METHYLATION IN LEUKEMIA .. 11 FLT3 LESIONS IN LEUKEMIA ................................................................................. 20 THE ROLES OF HISTONE POST-TRANSLATIONAL MODIFICATIONS IN ALTERNATIVE SPLICING .................................................................................................... 24 NON-CANONICAL PATHWAYS REGULATING GROWTH SIGNALING AND HISTONE METHYLATION MEDIATE THE SENSITIVITY OF MLL-REARRANGED LEUKEMIA TO LOW-DOSE DOT1L INHIBITION ................................................................. 31 INTRODUCTION ........................................................................................................ 31 RESULTS ..................................................................................................................... 34 2.2.1 MLL-r leukemia is sensitive to DOT1L inhibition via a non-canonical pathway .... 34 2.2.2 DOT1L inhibition at low concentrations downregulates leukemic oncogenes ........ 36 iii 2.2.3 MLL-AF4 targets downregulated by low dose DOT1L inhibition are highly enriched for H3K79me2 ...................................................................................................................... 44 2.2.4 MLL-r cells with FLT3-ITD mutations are hypersensitive to both FLT3 and DOT1L inhibition ............................................................................................................................... 50 2.2.5 Reduced FLT3 signaling by DOT1L inhibition culminates in reduced transcription of STAT5A target genes ....................................................................................................... 53 2.2.6 Overexpression of constitutively active STAT5A rescues proliferation and target gene expression defects caused by DOT1L inhibition ......................................................... 57 2.2.7 An ancillary DOT1L-dependent pathway limits proliferation through PRC2 signaling ................................................................................................................................ 61 2.2.8 STAT5A-CA overexpression rescues the viability of MV4;11 cells treated with MLL1 inhibitors .................................................................................................................... 65 DISCUSSION ............................................................................................................... 71 2.3.1 The FLT3-ITD signaling pathway accounts for the bulk of low-dose DOT1L inhibitor toxicity .................................................................................................................... 72 2.3.2 Extensive histone modification cross-talk contributes to the survival of MLL-r, FLT3-ITD+ leukemia ........................................................................................................... 75 2.3.3 Broader Clinical implications ................................................................................... 77 MATERIALS AND METHODS .................................................................................. 78 2.4.1 Accession numbers ................................................................................................... 78 2.4.2 Cell Culture ............................................................................................................... 78 2.4.3 RNA-seq and Gene Expression Analysis ................................................................. 79 2.4.4 Reverse Transcription and Quantitative real-time PCR ............................................ 80 iv 2.4.5 Cell Proliferation Assay ............................................................................................ 80 2.4.6 Apoptosis Assay ....................................................................................................... 80 2.4.7 Calibrated chromatin immunoprecipitation sequencing (ICeChIP-seq) ................... 81 2.4.8 Western blotting ........................................................................................................ 82 2.4.9 Plasmid generation .................................................................................................... 82 2.4.10 Transfection for lentiviral particle generation ...................................................... 83 2.4.11 Lentiviral transduction .......................................................................................... 83 DOT1L INHIBITION IMPACTS ALTERNATIVE SPLICING THROUGH RECRUITMENT OF THE SPLICING FACTOR PTBP1 ........................................................... 84 INTRODUCTION ........................................................................................................ 84 RESULTS ..................................................................................................................... 89 3.2.1 DOT1L inhibition results in greater exon inclusion of alternatively spliced genes . 89 3.2.2 Splicing factors including PTBP1 are enriched in H3K79me2-modified nucleosome pulldowns from nuclear extract ............................................................................................ 92 3.2.3 Pinometostat-induced alternatively spliced genes have aberrantly high H3K79me2 and show stark increases in H3K36me3 upon pinometostat treatment ................................ 94 3.2.4 PTBP1 knockdown reproduces pinometostat-induced effects on alternative splicing and reduces leukemia cell viability ....................................................................................... 99 3.2.5 H3K79me2 depletion results in alternative splicing of the PTEN tumor suppressor, increased PTEN protein levels and reduced AKT signaling .............................................. 101 DISCUSSION ............................................................................................................. 102 3.3.1 H3K79me2 is recognized by splicing factors including PTBP1, and depleting H3K79me2 or PTBP1 results in similar effects on splicing ............................................... 102 v 3.3.2 PTBP1 knockdown decreases MV4;11 MLL-r leukemia viability, consistent with its role in promoting proliferation in other malignancies ........................................................ 105 3.3.3 H3K79me2-regulated alternatively spliced genes have abnormally high H3K79me2 and large pinometostat-induced increases in H3K36me3 ................................................... 106 MATERIALS AND METHODS ................................................................................ 108 3.4.1 Accession numbers ................................................................................................. 108 3.4.2 Cell Culture ............................................................................................................. 108 3.4.3 Reverse Transcription and Quantitative real-time PCR .......................................... 109 3.4.4 Cell Proliferation Assay .......................................................................................... 109 3.4.5 Western blotting ...................................................................................................... 110 3.4.6 Plasmid generation .................................................................................................. 110 3.4.7 Transfection for lentiviral particle generation ........................................................ 110 3.4.8 Lentiviral transduction ............................................................................................ 111 3.4.9 Octamer assembly ................................................................................................... 111 3.4.10 Nucleosome reconstitution .................................................................................. 112 3.4.11 Nuclear extraction ............................................................................................... 112 3.4.12 Nucleosome pulldown ........................................................................................ 113 CONCLUSIONS ................................................................................................................ 115 MANY FUNCTIONS OF H3K79ME LACK MECHANISTIC EXPLANATION .. 115 THE FLT3-ITD/STAT5A SIGNALING PATHWAY IS ACTIVATED