Published OnlineFirst December 6, 2018; DOI: 10.1158/1535-7163.MCT-18-0395 Cancer Biology and Translational Studies Molecular Cancer Therapeutics KDM5B Promotes Drug Resistance by Regulating Melanoma-Propagating Cell Subpopulations Xiaoni Liu1,2, Shang-Min Zhang1, Meaghan K. McGeary1,2, Irina Krykbaeva1,2, Ling Lai3, Daniel J. Jansen4, Stephen C. Kales4, Anton Simeonov4, Matthew D. Hall4, Daniel P. Kelly3, Marcus W. Bosenberg1,2,5, and Qin Yan1 Abstract Tumor heterogeneity is a major challenge for cancer elevated KDM5B expression, melanoma cells shift toward a À treatment, especially due to the presence of various sub- more drug-tolerant, CD34 state upon exposure to BRAF populations with stem cell or progenitor cell properties. inhibitor or combined BRAF inhibitor and MEK inhibitor þ À þ In mouse melanomas, both CD34 p75 (CD34 )and treatment. KDM5B loss or inhibition shifts melanoma cells À À À þ CD34 p75 (CD34 ) tumor subpopulations were charac- to the more BRAF inhibitor–sensitive CD34 state. These terized as melanoma-propagating cells (MPC) that exhibit results support that KDM5B is a critical epigenetic regulator some of those key features. However, these two subpopula- that governs the transition of key MPC subpopulations tions differ from each other in tumorigenic potential, with distinct drug sensitivity. This study also emphasizes ability to recapitulate heterogeneity, and chemoresistance. the importance of continuing to advance our understand- þ À In this study, we demonstrate that CD34 and CD34 ing of intratumor heterogeneity and ultimately develop V600E subpopulations carrying the BRAF mutation confer novel therapeutics by altering the heterogeneous character- differential sensitivity to targeted BRAF inhibition. Through istics of melanoma. À þ þ CD34 p75 (p75 ), exhibit distinguishing cellular behaviors (8). Introduction þ Specifically, CD34 cells are highly tumorigenic in vivo, whereas fi þ À Intratumor heterogeneity serves as a signi cant clinical barrier p75 cells are rarely tumorigenic; CD34 cells exhibit an inter- fi to nding effective and long-lasting treatments for various types of mediate level of tumorigenic potential but are capable of reca- cancer (1). Many reports suggest that melanoma does not follow pitulating tumor heterogeneity in vitro and in vivo. Thus, both þ À the hierarchical cancer stem cell model because tumorigenic cells CD34 and CD34 cells are referred to as melanoma-propagating within a single melanoma tumor represent more than a rare cells (MPC) and are speculated to play a critical role in tumor – subpopulation (2 5). Meanwhile, several other studies argue that development. In addition, these three subpopulations demon- there exist more subtleties to this conclusion than previously strate varying levels of resistance to traditional cytotoxic che- suggested (6, 7). Our laboratory demonstrated that mouse motherapeutics in vitro, suggesting that they may also respond melanoma tumors driven by different genetic alterations are differently to more targeted treatments (8). Overall, although we composed of three major subpopulations. These three subpopu- þ À þ À À À have made progress in understanding the cellular characteristics lations, CD34 p75 (CD34 ), CD34 p75 (CD34 ), and representing the different tumor cell subpopulations in melano- ma, we have not yet identified key regulators mediating the þ À 1Department of Pathology, Yale School of Medicine, New Haven, Connecticut. conversion of CD34 and CD34 MPCs. This is an essential 2Department of Dermatology, Yale School of Medicine, New Haven, Connecticut. step to provide effective therapeutic strategies for melanoma 3Penn Cardiovascular Institute, Department of Medicine, Perelman School of patients considering the critical cellular features unique to these Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. 4National subpopulations. Center for Advancing Translational Sciences, National Institutes of Health, Several epigenetic regulators have been used to identify het- 5 Rockville, Maryland. Department of Immunobiology, Yale School of Medicine, erogeneous tumor subpopulations with distinct cellular pheno- New Haven, Connecticut. types. Non–small cell lung cancer cell lines along with several Note: Supplementary data for this article are available at Molecular Cancer other human cancer cell lines contained a reversibly drug-resistant Therapeutics Online (http://mct.aacrjournals.org/). subpopulation that required KDM5A to maintain the drug- X. Liu and S.M. Zhang contributed equally to this article. tolerant state (9–11). A slow-cycling subpopulation of melanoma M.W. Bosenberg and Q. Yan contributed equally to this article. cells expressing high levels of histone demethylase KDM5B (also Corresponding Authors: Qin Yan, Yale School of Medicine, 310 Cedar St., BML known as JARID1B) was shown to be important in maintaining 348C, PO Box 208023, New Haven, CT 06520-8023. Phone: 203-785-6672; melanoma growth and intrinsically resistant to various cancer þ À þ Fax: 203-785-2443; E-mail: [email protected]; and Marcus W. Bosenberg, Yale drugs (12, 13). Moreover, although CD34 , CD34 , and p75 School of Medicine, 15 York Street, LMP Ste 5038A, New Haven, CT 06510. cells have been identified in mouse melanoma tumors with Phone: 203-737-3484; Fax: 203-785-6869; E-mail: different genetic alterations, subpopulations expressing the [email protected] same distribution of markers exhibit similar progenitor cell doi: 10.1158/1535-7163.MCT-18-0395 properties (8). Taken together, these findings indicate that epi- þ Ó2018 American Association for Cancer Research. genetic variation likely exists between the two MPC and p75 706 Mol Cancer Ther; 18(3) March 2019 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst December 6, 2018; DOI: 10.1158/1535-7163.MCT-18-0395 KDM5B Regulates Melanoma-Propagating Cell Subpopulations subpopulations and may also be the explanation for their diverse Life Technologies). Cells were stained with rat anti-CD34 cellular characteristics. (ThermoFisher 14-0341-82) and rabbit anti-Nerve Growth Factor In addition, cancer cells exploit intratumor heterogeneity as a Receptor/p75 (Millipore Sigma AB1554) in PBS supplemented defense mechanism in response to drug therapy (9, 13, 14). This with 1% BSA (Sigma-Aldrich A9647) at 4C for 30 minutes. Cells highlights the clinical need to understand the regulation of were washed 3 times with fluorescence-activated cell sorting intratumor heterogeneity during tumor development and in the (FACS) buffer and stained with Brilliant Violet 421 Donkey context of therapeutic treatment. In melanoma, over 50% of anti-rabbit IgG (BioLegend 406410), Alexa Fluor 647 Goat patients harbor the V600 mutation in BRAF kinase. This particular anti-rat IgG (ThermoFisher A-21247), and propidium iodide genetic alteration leads to constitutive activation of the MAPK (BioLegend 421301) at 4C for 15 minutes. Cells were washed pathway and contributes to uncontrolled cell proliferation (15). 3 times, resuspended in PBS, and analyzed by BD LSRII Flow Unfortunately, resistance to targeted BRAF inhibitors (BRAFi) Cytometer. Plots were generated using FlowJo 9.9.6. To sort þ such as vemurafenib presents a continuous challenge in the clinic. CD34 cells, YUMM1.7 cells were stained using the same protocol Multiple resistance mechanisms have been described, including and subjected to single-cell sorting using BD FACSAria into a reactivation of the MAPK pathway, upregulation of the compen- 96-well plate. Individual cells were expanded and tested by flow þ þ À satory PI3K signaling, and nongenetic modifications (16, 17). In cytometry to ensure CD34 purity. CD34 and CD34 cells used þ À this study, we examine how CD34 and CD34 MPC subpopula- for the RT-qPCR experiment were sorted using the same staining tions respond to targeted therapies to better understand the link protocol but collected using 15 mL conical tubes using the BD between intratumor heterogeneity and drug response. We further FACSAria or Sony SH800 Cell Sorter. To analyze subpopulation demonstrate that histone demethylase KDM5B is a key regulator distribution in grafted tumors by flow cytometry, tumors of the conversion between MPC subpopulations of mouse were dissociated by finely mincing and incubating in DMEM/ melanoma. F12 (1:1) containing 1 mg/mL Collagenase/Dispase (Sigma- Aldrich 10269638001) and 1X DNase I (Qiagen 79254) at 37C for 30 minutes. Dissociated tumors were titrated and filtered Materials and Methods through a 70 mm filter to yield a single-cell suspension before Cell culture FACS staining. Cells were stained using a similar protocol Yale University Mouse Melanoma cell lines (YUMM1.1, described above with an additional anti-CD45 APC-Cy7 antibody YUMM1.3, YUMM1.7, YUMM1.9, YUMM2.1, YUMM3.3, (BioLegend 103115). YUMM3.4, and YUMM5.1) used in this study were characterized previously (18). Cells were maintained in DMEM/F12 (1:1) Western blot analysis media containing 10% heat-inactivated FBS, 1% penicillin Mouse cells were collected following digestion with 0.25% streptomycin, and 1% MEM nonessential amino acids (Gibco Trypsin-EDTA and lysed on ice with high salt lysis buffer by Life Technologies) at 37 Cand5%CO2. BRAFi-resistant [50 mmol/L Tris-HCl pH 7.9, 0.1 mmol/L EDTA pH 8.0, YUMM1.7 (YUMM1.7R) cells were derived by continuously 320 mmol/L NaCl, 0.5% NP40, 10% Glycerol, and 1x Protease exposing YUMM1.7 cells to 2 mmol/L BRAFi (PLX4032, BioVision Inhibitor cocktail (Roche