MERTK inhibition selectively activates a DC – T-cell axis to provide anti-leukemia immunity Restricted; Files Only

Abstract

TAM-family tyrosine kinases (TYRO3, AXL and MERTK) are potential cancer therapeutic targets in a wide range of human cancers. In previous studies, MERTK inhibition in the immune microenvironment was therapeutically effective in a B-cell acute leukemia (B-ALL) model. Here, we probed anti-leukemia immune mechanisms and evaluated roles for TYRO3 and AXL in the leukemia microenvironment. Host Mertk knockout or MERTK inhibitor MRX-2843 increased CD8α⁺ dendritic cells (DCs) with enhanced antigen-presentation capacity in the leukemia microenvironment and inhibited leukemogenesis. High MERTK or low DC gene expression were associated with poor prognosis in pediatric patients with ALL, indicating the clinical relevance of these findings. MRX-2843 also decreased potentially exhausted TOX^[HIGH] CD8⁺ T-cells, implicating a DC – T-cell axis. Indeed, combined depletion of CD8α⁺ DCs and CD8⁺ T-cells was required to abrogate anti-leukemia immunity in Mertk^–/– mice. Tyro3^–/– mice were also protected against B-ALL, implicating TYRO3 as an immunotherapeutic target. In contrast to Mertk^–/– mice, Tyro3^–/– did not impact CD8α⁺ DC frequency or antigen-presentation capacity and therapeutic activity was less dependent on DCs, indicating a different immune mechanism.

In contrast to Mertk^–/– or Tyro3^–/–, host Axl deletion did not impact B-ALL leukemogenesis. Moreover, host knockout of all three TAM-family kinases prolonged survival in an MML-AF9 AML model, while only Mertk ablation provided immunity in a second AML model, suggesting context-dependent roles of individual TAM-family kinases in anti-leukemia immunity.

These data demonstrate differential roles for TAM-family kinases in the leukemia microenvironment and provide rationale for the development of MERTK and/or TYRO3-targeted B-ALL immunotherapies.

Table of Contents

Distribution agreement I

Approval page. II

Abstract cover page. II

Abstract III

Cover page. IV

Acknowledgements. V

Table of contents. VII

List of abbreviations. X

List of figures. XVI

Chapter 1: Introduction. 1

1.1 Acute leukemia. 1

1.1.1    Pathobiology of acute leukemia. 1

1.1.2 Current standard of care in pediatric patients. 2

1.1.3 Challenges of current cytotoxic chemotherapies. 3

1.2 Novel treatment approaches for acute leukemia. 5

1.2.1 Targeted therapies in acute leukemia. 5

1.2.2 The acute leukemia immune microenvironment 6

1.2.3 Immunotherapies in acute leukemia. 8

1.3 TAM family RTKs as therapeutic cancer targets. 12

1.3.1 Introduction to TAM RTKs. 12

1.3.2 Physiologic TAM RTK functions. 15

1.3.3 Targeting TAM RTKs in cancer cells. 20

1.3.4 Pathologic roles for TAM RTKs of the TME in suppressing anti-cancer immunity. 23

1.3.5 TAM RTKs as immunotherapeutic targets in acute leukemia. 29

1.4 Aims and approach. 31

Chapter 2. MERTK inhibition selectively activates a DC – T-cell axis to provide anti-leukemia immunity in B cell acute leukemia. 33

2.1 Introduction. 33

2.2 Materials and methods. 35

2.2.1 Cell culture. 35

2.2.2 Murine B-cell ALL model 35

2.2.3 Immune cell depletion. 35

2.2.4 Murine tissue collection. 36

2.2.5 Single cell RNA sequencing and data processing. 36

2.2.6 Pathway and systems biology analysis. 38

2.2.7 Flow cytometry. 38

2.2.8 TARGET survival data. 40

2.2.9 Statistics. 40

2.3 Results. 41

2.3.1 MERTK inhibitor MRX-2843 increases immune cells in the leukemia microenvironment and promotes anti-leukemia immunity. 41

2.3.2 TAM-family kinases have differential roles in the leukemia microenvironment. 46

2.3.3 Mertk–/– or MRX-2843 but not Tyro3–/– increases CD8α+ DCs with markers indicating increased antigen-presenting capacity. 50

2.3.4 Mertk knockout increases CADM1 and Cathepsin E expressing DCs. 59

2.3.5 MRX-2843 reduces T-cell exhaustion markers in the leukemia microenvironment. 61

2.3.6 NK1.1+ cells are not required for anti-leukemia immunity in Mertk–/– mice. 62

2.3.7 Combined depletion of CD8α+ DCs and CD8+ T-cells is required to abrogate anti-tumor immunity in Mertk–/– mice. 67

2.3.8 Low MERTK expression or high DC gene set expression are associated with improved survival in patients with high-risk pediatric ALL. 68

2.4 Discussion. 74

Chapter 3. TAM RTKs have differential roles in suppressing anti-AML immunity depending on disease model 77

3.1 Introduction. 77

3.2 Material and Methods. 78

3.2.1 Cell culture. 78

3.2.2 AML mouse models. 78

3.2.3 Statistics. 78

3.3 Results. 79

3.3.1 Host ablation of all three TAM RTKs prolongs survival in MML-AF9 AML model, while only Mertk ablation provides immunity in a second AML model. 79

3.4 Discussion. 80

Chapter 4: Conclusions and closing remarks. 81

4.1 Summary. 81

4.2 Differential roles for TAM RTKs in the acute leukemia microenvironment 82

4.3 Further implications for the development of TAM-targeted acute leukemia therapies. 85

4.4 Future directions. 88

References. 90

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Cancer Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Cell
Keyword	Dendritic Cells Receptor Tyrosine Kinases Cancer Immunology Acute Leukemia Immunotherapies TAM RTKs Targeted Therapies
Committee Chair / Thesis Advisor	Douglas Graham, Emory University
Committee Members	Nicki Panoskaltsis, Trinity College Dublin, Ireland Haydn Kissick, Emory University Gregory Lesinski, Emory University Curtis Henry, University of Colorado

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