Imetelstat Inhibits Telomerase and Prevents Propagation of ADAR1- activated Myeloproliferative Neoplasm and Leukemia Stem Cells

Wenxue Ma1, Larisa Balaian1, Phoebe Mondala1, Yudou He1, Cayla Mason1, Jessica Pham1, Jeremy Lee1, Raymond Diep1, Sanja Coso1, Kathleen Fisch1, Adam Mark1, Sheldon Morris1, Qingfei Jiang1, Thomas Whisenant1, Aleksandra Rizo2, Fei Huang2, Mary Donohoe1, Ludmil Alexandrov1, and Catriona Jamieson1*

1 Division of Regenerative Medicine, Department of Medicine, Moores Cancer Center, and Sanford Stem Cell Clinical Center, University of California San Diego, La Jolla, CA; 2 Geron Corporation, Parsippany, NJ.

*Corresponding author: Catriona Jamieson, MD PhD, Deputy Director, UC San Diego Moores Cancer Center and Director, Sanford Stem Cell Clinical Center, [email protected] Fig. 1 Telomere Shortening Characterizes Pre-LSC and LSC

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Whole genome sequencing revealed significant telomere shortening in stem cells during myeloproliferative neoplasm (MPN) progression Fig. 2 Pre-LSC and LSC Harbor Increased TERT and ADAR1 Expression

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C RNA-seq showed that ADAR1p150 increased in progenitors during MPN progression, which co- localized with TERT in TF1a leukemia cells, while lentiviral shRNA knockdown reduced ADAR1 and hTERT expression as shown by confocal fluorescence microscopy. Fig. 3 Imetelstat Prevents Pre-LSC and LSC Maintenance In vitro

+ A Dividing stromal Non-proliferating CD34 cells selected cell lines (human SCF, IL-3, G-CSF from BC CML, MF or (SL and M2) producing) stromal cells control aNBM Imetelstat, a SL Irradiation competitive inhibitor SLM2 Stroma M2 of telomerase Imelestat +/- Dasatinib (Pacritinib) enzymatic activity, at the initiation of co-culture Survival Self-renewal selectively inhibited myelofibrosis (MF) qRT-PCR for Co-culture gene expression for 7-14 days stem cell survival and self-renewal in vitro,

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Imetelstat reduced MF progenitor engraftment in a humanized NSG-SGM3 mouse model and serial transplantation of human LSC engraftment in BC CML models. Fig. 5 Imetelstat Prevents ADAR1-mediated RNA Editing in LSC

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Human BC CML LSC eradication by imetelstat in mouse models was associated with decreased hTERT, D E F telomerase activity, beta- catenin activity, ABL expression and ADAR1 activation Conclusions

1. Whole genome sequencing revealed significant telomere shortening in stem cells during myeloproliferative neoplasm (MPN) progression. 2. Whole transcriptome sequencing (RNA-seq) revealed an increase in telomerase reverse transcriptase (hTERT) 3. RNA-seq showed that ADAR1p150 increased in progenitors during MPN progression, which co-localized with TERT in TF1a leukemia cells, while lentiviral shRNA knockdown reduced ADAR1 and hTERT expression as shown by confocal fluorescence microscopy. 4. Imetelstat, a competitive inhibitor of telomerase enzymatic activity, selectively inhibited myelofibrosis (MF) stem cell survival and self-renewal in vitro, while combination of imetelstat with dasatinib was required to inhibit leukemia stem cell survival and self-renewal in blast crisis chronic myeloid leukemia (BC CML) 5. Imetelstat also reduced MF progenitor engraftment in a humanized NSG-SGM mouse model and serial transplantation of human LSC engraftment in blast crisis BC CML models. 6. Human BC CML LSC eradication by imetelstat in mouse models was associated with decreased hTERT, telomerase activity, beta-catenin activity, ABL expression and ADAR1 activation 7. These data suggest that Imetelstat may prevent LSC-driven blast crisis transformation.

Funding sources: MPN Challenge Grant, LLS Blood Cancer Discoveries Grant, NASA NRA #: NNJ13ZBG001N, NIH NIDDK R01 DK114468 , NIH NCI R01CA205944 , Moores Family Foundation, Koman Family Foundation, Sanford Stem Cell Clinical Center, J&J