Identification and characterization of novel genetic and epigenetic factors required for normal and malignant hematopoiesis

Acute myeloid leukemia (AML) is a type of blood cancer, characterized by clonal expansion and loss of differentiation ability of myeloid progenitor cells leading to abnormal accumulation of immature myeloid cells (myeloblasts) in the bone marrow and peripheral blood. This thesis (study I to IV) focused on the identification and characterization of genes which are required for AML growth. The final study (study V) aimed to uncover the role of NAP1L3 in normal hematopoietic stem cells (HSCs).

In studies I and II, we performed large-scale RNA interference screens in mouse and AML human cell lines to identify novel factors and pathways required for AML growth. Using this approach, we identified two novel targets: Chromatin remodeling factor CHD4 (study I) and the transcription factor GTF2IRD1 (study II), which display both a strong inhibitory effect on the growth of AML cells and a less negative effect on normal hematopoietic cells. Using RNA interference and CRISPR-Cas9 techniques, we revealed that these genes were crucial for AML cell growth in vitro and in vivo. Knockdown of either CHD4 or GTF2IRD1 accumulated cells in the G0 phase of the cell cycle and resulted in downregulation of MYC and its target genes. We demonstrated the inhibitory role of CHD4 knockdown on the growth and maintenance of primary childhood AML in an ex vivo setting, as well as in a xenograft model by transplanting patient-derived samples into humanized NSG-SGM3 mice. GTF2IRD1 knockdown reduced the number of primary childhood and adult AML cells in ex vivo culture and delayed AML progression in the transplanted animal model. Therefore, CHD4 and GTF2IRD1 are important for AML cell growth, and interestingly the knockdown of these two genes did not show a strong inhibitory effect on normal hematopoietic cell growth.

In study III, we described the role of an epigenetic enzyme, the histone methyl-transferase EHMT1 in AML. We used RNA interference, CRISPR-Cas9, and pharmacological approaches to inhibit EHMT1 expression, which prevented the growth of various AML cell lines and primary AML patient samples. Knockdown of EHMT1 significantly delayed disease progression in AML mouse models and prolonged their survival. Next, we employed CRISPR-Cas9 technology to generate single and double gene knockouts of EHMT1 and its homolog EHMT2, which showed that both enzymes cooperatively play a role in AML cell proliferation and shared a similar cellular mechanism as individual knockouts of either gene resulted in an increased number of cells in G0 phase of the cell cycle. RNA sequencing of the transcriptome of AML cells with EHMT1 and EHMT2 knockdown identified several common biological processes, including cell differentiation, proliferation and survival, as well as other unshared pathways and downstream effectors.

In study IV, we contributed to Nikolas Herold’s study, who found that deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAM domain and HD domain 1 (SAMHD1) plays a role in detoxifying intracellular ara-CTP in cells treated with the deoxycytidine analog cytarabine (ara-C). Transient reduction of SAMHD1 expression by using the simian immunodeficiency virus (SIV) protein Vpx significantly increased the sensitivity of AML cells to ara-C, whereas AML cells lacking SAMHD1 transplanted into recipient mice were hypersensitive to ara-C. We showed that in vitro treatment of primary AML patient samples with Vpx, which suppresses SAMHD1, resulted in reduced proliferation of AML but not normal cells. Together, our data suggest that SAMHD1 inhibition can be used as a therapeutic strategy for cancer (AML) patients with high SAMHD1 expression.

In study V, our aim was to identify novel epigenetic regulators of normal HSCs. We found high expression level of Nap1l3, a member of nucleosome assembly proteins (NAPs), as a histone chaperone in HSCs. Loss of function of mouse Nap1l3 mediated by shRNA or CRISPR-Cas9 impaired the maintenance and differentiation of HSCs in both our in vitro and in vivo studies. Moreover, downregulation of NAP1L3 in human UCB HSCs significantly decreased both the number of colonies formed by HSCs and their proliferation in vitro due to cell cycle arrest in the G0 phase. Xenograft mouse models using human HSCs with NAP1L3 knockdown showed a reduction of HSC reconstitution and bias in differentiation. Furthermore, we observed upregulation of several HOX genes (HOXA3, HOXA5, HOXA6 and HOXA9) under NAP1L3 suppression in human HSCs.

Altogether, in this thesis, we showed the important roles of CHD4, EHMT1 and GTF2IRD1 in AML cell expansion, identifying them as potential novel targets for AML treatment. Moreover, we revealed the cellular mechanisms and RNA expression patterns under knockdown of these genes. We contributed to the study that found SAMHD1 expression level can be used as a prognostic marker for ara-C treatment and that inhibition of SAMHD1 increases the sensitivity of AML cells to ara-C treatment. Finally, we identified a novel regulatory role for NAP1L3 as a histone chaperone in self-renewal and differentiation of HSCs.

List of scientific papers

I. The chromatin-remodeling factor CHD4 is required for maintenance of childhood acute myeloid leukemia. Yaser Heshmati, Gözde Türköz, Aditya Harisankar, Shabnam Kharazi, Johan Boström, Esmat Kamali Dolatabadi, Aleksandra Krstic, David Chang, Robert Månsson, Mikael Altun, Hong Qian and Julian Walfridsson. Haematologica. 2018, volume 103 (7), 1169-1181.
https://doi.org/10.3324/haematol.2017.183970

II. Identification of GTF2IRD1 as a novel transcription factor essential for acute myeloid leukemia. Yaser Heshmati, Gözde Türköz, Marios Dimitriou, Aditya Harisankar, Johan Boström, Mikael Altun, Hong Qian, Nadir Kadri, Julian Walfridsson. [Manuscript]

III. The histone methyltransferase EHMT1 plays both independent and cooperative regulatory role in the maintenance of Acute Myeloid Leukemia. Yaser Heshmati, Gözde Türköz, Emma Wagner, Aditya Harisankar, Johan Boström, Mikael Altun, Hong Qian and Julian Walfridsson. [Manuscript]

IV. Targeting SAMHD1 with the Vpx protein to improve cytarabine therapy for hematological malignancies. Nikolas Herold, Sean G Rudd, Linda Ljungblad, Kumar Sanjiv, Ida Hed Myrberg, Cynthia B J Paulin, Yaser Heshmati, Anna Hagenkort, Juliane Kutzner, Brent D G Page, José M Calderón-Montaño, Olga Loseva, Ann-Sofie Jemth, Lorenzo Bulli, Hanna Axelsson, Bianca Tesi, Nicholas C K Valerie, Andreas Höglund, Julia Bladh, Elisée Wiita, Mikael Sundin, Michael Uhlin, Georgios Rassidakis, Mats Heyman, Katja Pokrovskaja Tamm, Ulrika Warpman-Berglund, Julian Walfridsson, Sören Lehmann, Dan Grandér, Thomas Lundbäck, Per Kogner1, Jan-Inge Henter, Thomas Helleday & Torsten Schaller. Nature Medicine. 2017, Volume 23, 252-263.
https://doi.org/10.1038/nm.4265

V. The histone chaperone NAP1L3 is required for hematopoietic stem cell maintenance and differentiation. Yaser Heshmati, Shabnam Kharazi, Gözde Türköz, David Chang, Esmat Kamali Dolatabadi, Johan Boström, Aleksandra Krstic, Theodora Boukoura, Emma Wagner, Nadir Kadri, Robert Månsson, Mikael Altun, Hong Qian, Julian Walfridsson. Scientific reports. 2018, volume 8(1)11202.
https://doi.org/10.1038/s41598-018-29518-z