Page 174 - Haematologica March 2020

C. Annageldiyev et al.
tial in animal models.4,6-8 After relapse, numbers of LSC increase dramatically and CD34– cells often acquire engraftment potential.6,9
Inclusion of additional AML-specific LSC surface anti- gens, including CD123, CD96 and TIM-3, can help identi- fy and target resistant leukemic cells.10-13 It has been sug- gested that the self-renewal capacity of otherwise quies- cent AML-LSC is supported by upregulation of the surface marker T-cell immunoglobulin mucin-3 (TIM-3). TIM-3 is not expressed in normal HSC, suggesting that the TIM-3+ population may contain the great majority of functional LSC in most types of AML.14 These markers play a role in activating the inactive LSC for the purpose of self-renewal and disease maintenance, thus facilitating relapse with minimal to moderate survival benefit.12-16
Stem cells protect themselves by upregulation of alde- hyde dehydrogenase (ALDH), a cytosolic enzyme that guards them against the DNA damage induced by reactive oxygen species and reactive aldehydes.17 A population of CD34+CD38− leukemic cells with moderate ALDH activi- ty has been shown to contribute to relapse in AML.18 Targeting intracellular markers including ALDH and signal transducer and activator of transcription 3 (STAT3) in LSC marked by additional surface markers like CD34, CD123, TIM-3 or CD96 may validate therapeutic targets more efficiently. Despite substantial advances in the under- standing of LSC markers, so far, no agents have been made available in the clinic to selectively target these pro- genitors. Cytarabine (Ara-C) and anthracyclines (7+3) are the current standard induction and consolidation therapy for AML, but these regimes only provide moderate thera- peutic benefit.19 The recent approval of novel agents including venetoclax, gilteritinib, and midostaurin has advanced therapy.
In this study, we identify the unexplored anti-LSC activ- ity of the recently published small molecule Isatin analog, KS99. Earlier studies had established KS99 as an anti- microtubule agent with a dual role as Bruton’s tyrosine kinase (BTK) inhibitor in multiple myeloma (MM).20 Since BTK has a role in the maturation and regulation of den- dritic cells (DC) via interleukin 10 (IL-10) and Signal trans- ducer and activator of transcription 3 (STAT3), blocking BTK carefully modulates the STAT3.21 Modulation of STAT3 is important in prolonging survival of AML patients, especially considering that upstream mutations result in the activation of STAT3 and the protein per se is not mutated in this condition.22 STAT3 activity in LSC is associated with a poor prognosis in AML patients, possi- bly because it contributes to resistance to chemothera- py.22,23 ALDH has been identified as a potential biomarker and therapeutic target in chemoresistant AML.24-26 Here, we report that, besides BTK inhibition, KS99 targets stem- ness markers, STAT3, and ALDH, in putative LSC express- ing surface CD34, CD123, TIM-3, and CD96. We demon- strate that KS99 is active against AML as a single agent or in combination with standard of care Ara-C.
Methods
The Online Supplementary Appendix contains detailed informa- tion on experimental methods and materials.
Cell lines and cell culture
Details of the acute myeloid leukemia cell line culture condi- tions are provided in the Online Supplementary Appendix.
Acute myeloid leukemia patient and healthy donor cells
Bone marrow (BM) aspirates or peripheral blood (PB) samples were obtained from AML patients, and cord blood (CB) samples were obtained from the freshly delivered placenta of healthy donors after informed consent using protocols approved by the Penn State College of Medicine Institutional Review Board (IRB) (#2000-186). Mononuclear cells (MNC) were isolated by density gradient separation (Ficol-Paque, GE Healthcare Life Sciences, Pittsburgh, PA, USA) and frozen for later use. Details are provided in the Online Supplementary Appendix.
Cell viability and Annexin V assay
Cell viability and apoptosis were determined using MTS [3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul- fophenyl)-2H-tetrazolium, inner salt] assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA) and Muse Annexin V & Dead Cell Kit (MCH100105, Millipore, Burlington, MA, USA). Details are provided in the Online Supplementary Appendix.
Colony-forming assay
Cryopreserved human AML patient samples and cord blood mononuclear cells were thawed and washed with RPMI 1640 (10% FBS) and used for the colony formation assay. Details are provided in the Online Supplementary Appendix.
Western blot analysis
Acute myeloid leukemia cells were treated with indicated con- centrations of KS99 or DMSO. Cells were collected, washed with cold PBS, and whole cell lysates were harvested. Further details are provided in the Online Supplementary Appendix.
Flow cytometry
To detect apoptosis in LSC, DMSO, KS99 or Ara-C-treated cells were washed and stained with various markers; anti-human CD45 conjugated with APC-Cy7, CD34-FITC, CD38-APC, CD123-APC, TIM-3-PE-Cy7, or CD96-BV711 monoclonal anti- bodies for 30 minutes on ice, followed by Annexin V-BV421 and 7AAD staining. Further details are provided in the Online Supplementary Appendix.
Aldehyde dehydrogenase assay
The enzyme activity of ALDH was measured by using ALDE- FLUOR kit (StemCell Technologies, Vancouver, Canada), as described in the manufacturer's protocol. It is a fluorescent-based assay that detects ALDH1A1 isoform, which is highly expressed in stem cells. Further details are provided in the Online Supplementary Appendix.
Animal studies
Acute myeloid leukemia cell transplantable models luciferase- expressing human AML cell lines (U937 and MV4-11) and murine AML cell line (C1498) were used to investigate the efficacy of KS99. In addition, the pharmacokinetics of the drug were exam- ined to determine the circulating levels of KS99 in the blood. Further details are provided in the Online Supplementary Appendix.
In silico docking of KS99 with ALDH1A1, BTK, and STAT3
Details are provided in the Online Supplementary Appendix.
Statistical analysis
The statistical analysis methodology is described in the Online Supplementary Appendix.
688
haematologica | 2020; 105(3)