Targeting Cathepsin-G to Overcome Leukemic Stem Cell Persistence in Chronic Myeloid

Targeting Cathepsin-G to Overcome Leukemic Stem Cell Persistence in Chronic Myeloid | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Targeting Cathepsin-G to Overcome Leukemic Stem Cell Persistence in Chronic Myeloid Esther Sathya Bama Benjamin, Raveen Stephen Stallon Illangeswaran, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8580579/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract Chronic myeloid leukemia (CML) stem and progenitor cells (LSPCs) are not eliminated by tyrosine kinase inhibitors, resulting in disease relapse or progression. Transcriptomic profiling identified Cathepsin G ( CTSG ), a serine protease, as one of the top significantly upregulated targets in primary CML CD34 + cells compared to CD34 + cells from healthy donors (Log2 FC, p-value). Further, molecular inhibition of CTSG reduced the self-renewal capacity and diminished disease burden in vivo in a CDX mouse model, highlighting CTSG as a promising therapeutic target in CML. Pharmacological inhibition of CTSG demonstrated a dose-dependent decrease in CTSG expression and colony-forming capacity only in CML CD34 + cells. This study offers comprehensive insights into the gene expression landscape of CML LSPCs, highlighting the functional significance of CTSG in disease progression. Targeting CTSG could be a novel therapeutic strategy for CML treatment by disrupting the leukemogenic potential and improving therapeutic efficacy. Biological sciences/Cancer Biological sciences/Cell biology Biological sciences/Drug discovery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Tyrosine kinase inhibitors (TKIs) have revolutionized the management of chronic myeloid leukemia (CML) by significantly improving treatment outcomes. However, resistance to TKIs remains a challenge in some patients. While mutations in the BCR-ABL1 kinase domain explain resistance in certain cases, the cause of resistance remains unclear in the majority of patients. Emerging evidence suggests that leukemic stem cells (LSCs) may play a critical role in therapy resistance by evading TKI-mediated targeting through BCR-ABL1 -independent mechanisms. Unlike bulk leukemic cells, LSCs exhibit intrinsic resistance to TKIs ( 1 , 2 ). Understanding the molecular mechanisms underlying LSC resistance could aid in the development of targeted therapeutic strategies to selectively eliminate these cells. One approach involves comparing transcriptomic profiles between healthy and CML stem cells to identify specific gene signatures unique to CML stem cells. In the absence of definitive cell surface markers distinguishing CML LSCs from normal hematopoietic stem cells (HSCs), CD34 + cells are often considered a marker for leukemic stem and progenitor cells (LSPCs) in many studies. Despite efforts to characterize differences between CML cells and normal HSCs, only a few studies have employed CML CD34 + cells to identify these distinct signatures ( 3 – 8 ). Interestingly, the metabolic profiling of CML CD34 + cells has revealed distinct metabolic differences, highlighting enhanced oxidative phosphorylation (OxPHOS) when compared to healthy HSCs ( 9 ). Targeting these metabolic vulnerabilities has shown promise in preclinical studies. For instance, tigecycline, an antibiotic, also effectively inhibited OxPHOS in treatment-resistant CML LSCs, both in vitro and in vivo, while sparing normal HSCs. However, in a Phase I trial involving patients with relapsed/refractory AML, tigecycline demonstrated a satisfactory safety profile but failed to yield significant clinical benefits, likely due to its shorter half-life ( 10 ). These findings underscore the need to explore alternative strategies for targeting LSCs. Previous reports on transcriptomic signatures in CML have highlighted an increased expression of focal adhesion-related genes in CML CD34 + cells and identified Integrin-linked kinase (ILK) as a crucial focal adhesion component required for LSC self-renewal in vivo. The study predominantly utilized in vitro and in vivo models to assess the effects of ILK inhibition ( 11 ). Similarly, Zhao et al. (2022) reported the role of MS4A3 (Membrane spanning 4-domains A3) as a functional tumor suppressor in CML, where its downregulation resulted in the retention of leukemia stem/progenitor cells (LSPCs) in a state of differentiation blockage that rendered them insensitive to TKIs. In this recent study, exogenous MS4A3 protein was introduced using a targeted delivery strategy, although the feasibility of clinical translation for this approach remains uncertain ( 12 ). Overall, previous studies highlight the significance of considering metabolic differences and alternative signaling pathways in unraveling treatment-resistant cells. In the present study, using a global transcriptomics approach, we aimed to identify gene signatures and pathways differentially expressed in CML CD34 + cells to better understand the mechanisms contributing to CML LSC survival. Materials and Methods Ethics and IRB statement All the study procedures and methods were performed in accordance with the Declaration of Helsinki and Institutional guidelines. The experimental protocols were reviewed and approved by institutional review Board (IRB), Christian Medical College, Vellore (IRB Min No. 14754 dated 27.07.2022). Informed Consent was obtained from all the participants prior to inclusion in this study. Primary cells and Cell lines After obtaining written informed consent, Peripheral blood / Bone marrow (BM) aspirates were collected from patients with chronic phase CML at diagnosis. The experimental protocols were reviewed and approved by institutional review Board (IRB), Christian Medical College, Vellore (IRB Min No. 14754 dated 27.07.2022). Informed Consent was obtained from all the participants prior to inclusion in this study. EM-2, KU812, Lama84, KCL22, KYO, MEG01, JURL-MK1, and K562 cell lines were procured from DSMZ, German Collection of Microorganisms and Cell Cultures. All the cell lines were maintained in RPMI supplemented with 10% Fetal Bovine Serum (Gibco) and 1% Penicillin and streptomycin (Gibco). All cell lines were periodically tested for mycoplasma (Universal Mycoplasma Detection Kit, ATCC) and were mycoplasma-free. Isolation and culture of CD34 + cells Briefly, Mononuclear cells (MNCs) were isolated using Lymphoprep (STEMCELL Technologies) by density gradient separation, and CD34 + cells were enriched immunomagnetically using an EasySep CD34 Positive Selection Kit (STEMCELL Technologies) following the manufacturer's protocol. Purity was verified by re-staining isolated cells with an allophycocyanin-labeled (APC) anti-CD34 antibody (Biolegend) and analyzing cells on a FACS Accuri C6 (BD) instrument. Cryopreserved CD34 + cells of the CML patients and the normal donor were thawed and cultured in StemSpan™ SFEM II medium (Stem Cell Technologies) supplemented with CD34 + Expansion Supplement (Stem Cell Technologies) and UM729 (Stem Cell Technologies). The cells were cultured for 6–7 days with half-medium change every other day to assess the cell proliferation. cDNA synthesis Total RNA was extracted from PB/BM Cells using TRI reagent (Sigma, St. Louis, USA) per the manufacturer's protocol. Following the manufacturer's instructions, 2µg of total RNA was converted to cDNA using a High-Capacity Reverse Transcription Kit (ThermoFisher Scientific) and stored at -20°C for further downstream applications. Confirmation of cDNA conversion was done using RT-PCR to test the expression of housekeeping gene β-glucuronidase ( GUS ) (Table 3.2). Strand-specific RNA-sequencing RNA was extracted from CML CD34 + (N = 10) and mobilized peripheral blood (mPB) CD34 + (N = 4) cells using Total RNA Purification Plus Kit (Norgen Biotek) as described earlier and then subjected to Strand-specific transcriptomics. RNA QC was evaluated using Agilent Bioanalyzer 2100 and Qubit High sensitivity RNA assay. The transcriptome profile of CML CD34 + cells was compared to the mPB CD34 + cells. The raw reads were filtered using Trimmomatic for quality scores and adapters. The filtered reads were aligned to the Human genome (hg19) using splice-aware aligner like HISAT2 to quantify reads mapped to each transcript. The alignment percentage of the reads was in the range of 5.5–97.5% for all the samples. The total number of uniquely mapped reads was counted using feature counts. The uniquely mapped reads were then subjected to differential gene expression using Deseq2 (Figure S1). The datasets generated and/or analysed during the current study are available in the gene expression omnibus (GEO) repository, GSE315681. NanoString nCounter Plexset custom panel Based on the differentially expressed genes list obtained from the transcriptomic data, we custom-designed a gene panel consisting of 21 target genes associated with cell surface markers, transporters, cell adhesion, and immune regulation based on previous publications (Supplementary Table 2). The expression of these 21 genes was then examined in an independent cohort of CML CD34 + (N = 39) and mPB CD34 + (N = 9) cells. Read counts were normalized using positive control and three housekeeping genes ( ACTB , GAPDH , and GUSB ). Immunoblot Protein lysates were prepared using radioimmunoprecipitation assay buffer supplemented with a protease inhibitor mixture and phenylmethylsulfonylfluoride. 30 µg of the cell lysate was loaded onto a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with non-fat dry milk powder in tris-buffered saline containing Tween 20 and incubated with primary antibodies for CTSG and β-actin. The proteins were detected using the ECL chemiluminescence kit and imaged using the FluorChem E system ( 13 ). Lentiviral-mediated CTSG knockdown in CML cells EM-2 cells were seeded at 2x10 5 cells/24-well and infected with 20ul of the concentrated lentiviral vector containing Doxycyline inducible shRNA constructs targeting human CTSG or a non-targeting sequence (Tet-pLKO-puro (Addgene: 21915)) in the presence of polybrene for 24 hours. The expression of the shRNA was induced with doxycycline (Dox) (1µg/ml) for 5 days; subsequently, western blotting was performed to confirm the knockdown ( 13 ). Colony-forming unit assay Briefly, 1,000 cells were mixed in 1 mL MethoCult (STEMCELL Technologies), and then the number of colonies was scored on day 14 following the manufacturer's protocol. Cell line-derived Xenograft model EM-2 cells were transduced with lentivirus containing the Dox inducible shCTSG, followed by Dox induction for 48h. These cells induced with and without dox were transplanted into sub-lethally irradiated (2 Gy) 8–10 weeks old NSG mice (N = 9 each) (The Jackson Laboratory, Bar Harbor, ME, USA) via the tail vein. Doxycycline was provided in drinking water at a concentration of 1mg/ml. The median survival of mice was evaluated. The difference in percentage survival between the CTSG WT and KD mice was evaluated by Kaplan-Meier survival analysis, and the median survival was compared using the Mann-Whitney U test. Results Transcriptome profiling in primary CML CD34 + cells reveals unique gene signatures compared to normal CD34 + cells To identify the unique gene signature of CML LSPCs, we conducted transcriptomic profiling of CML CD34 + and mPB CD34 + cells using NGS on an Illumina sequencing platform. Global transcriptome profiling of differentially expressed genes was evaluated by filtering based on the reads per kilobase million (RPKM) > 1 and a minimum of a 2-fold difference (CuffDiff, FDR-adjusted p < 0.05) between CML CD34 + and mPB CD34 + cells. Principal Component Analysis (PCA) revealed a distinct separation between the two groups, indicating an apparent clustering of samples that aligned with their respective groups. (Fig. 1A). We observed 1563 genes upregulated and 1400 genes downregulated in CML CD34 + cells compared to mPB CD34 + (Fig. 1B). Top upregulated genes include GSTM1 (Glutathione S-transferase mu 1), AZU1 (Azurocidin 1), LTF (Lactotransferrin ) , CTSG (Cathepsin G), HIST14C (histone proteins) and downregulated genes include CXCL13 (C-X-C ligand 13), ITGA3 (Integrin Subunit Alpha 3), CDH4 (Cadherin 4), PRMD16 (protein arginine methyltransferase 16) (Fig. 1C). Pathway enrichment analysis revealed that the upregulated genes are involved in immune response, cytokine signaling, and cell adhesion. Pathway enrichment analysis of the top differentially expressed genes (log 2 F > 1, p < 0.05) was done using Enrichr ( 11 ) ( https://maayanlab.cloud/Enrichr/ ). Based on Reactome, KEGG, and MSigDB datasets, genes associated with the cell cycle, Fanconi anemia pathway, and metabolism were significantly upregulated in CML CD34 + cells (Fig. 1D-E and S2A ). Gene ontology analysis using GO biological process, GO cellular component, and GO molecular function revealed that genes associated with mitotic sister chromatid segregation spindle, single-strand helicase activity, and mitochondrial matrix were dysregulated in CML CD34 + cells (Fig. 1F and S2B-C ). Pathway enrichment of significantly downregulated genes revealed the genes associated with cytokine signaling, inflammatory response, and TNFα signaling to be downregulated in CML CD34 + cells (Fig. 1G-H and S2D ). Together, these results suggest that in CML LSPCs, immune response, inflammation, and cytokine signaling were downregulated. Gene ontology analysis of significantly downregulated genes revealed the genes associated with positive regulation of cytokine production, Focal adhesion, and inhibitory MHCI receptor activity to be downregulated (Fig. 1I and S2E-F) . LSPCs- Specific gene signature Leukemic stem/progenitor cell (LSPC)-specific gene signature in chronic myeloid leukemia (CML) was evaluated by comparing transcriptomic data generated in this study with two publicly available CML CD34 + datasets ( 14 , 15 ). This integrative analysis was done to reduce dataset-specific biases and to identify robust, reproducible gene expression changes characteristic of CML LSPCs. Consistent upregulation of genes involved in heme metabolism, porphyrin metabolism, and heme biosynthesis pathways was observed across datasets (Fig. 2A-B ). These pathways are crucial for iron homeostasis and redox regulation, processes essential for cellular proliferation and survival in leukemic progenitors. In contrast, genes commonly downregulated were enriched for biological processes related to cellular and leukocyte homeostasis (Fig. 2C), indicating disruption of normal hematopoietic regulatory mechanisms. A core set of 28 genes was identified as consistently upregulated, while 18 genes were consistently downregulated across all datasets (Fig. 2D), implicating dysregulated iron metabolism as a key feature of CML LSPCs. Transcriptome analysis predicted unique cell surface marker expression in CML CD34 + cells: We then evaluated alterations in cell surface markers in CML CD34 + cells, as these changes can serve as biomarkers and help distinguish between LSPCs and HSCs. Our analysis revealed that in CML CD34 + cells, the expressions of CD200R1, CD276, CD96, and CD36 was increased, while CD79A, CD37 , and CD22 decreased compared to mPB donor CD34 + cells. While CD36 has been previously reported as a CML LSC marker, the upregulation of CD96, a recognized marker for AML LSCs, is noteworthy and requires further validation to distinguish CML LSPCs from HD HSPCs. ( Figure S3A ). CML CD34 + cells showed significantly increased expression of serine protease Cathepsin G: Among the significantly upregulated genes in CML CD34 + cells, one of the top targets was the serine protease family. Notably, serine proteases such as Cathepsin G ( CTSG) , Cathepsin L ( CTSL) , and neutrophil elastase ( ELANE ) were significantly upregulated in CML CD34 + cells. In addition, ribonuclease ( RNASE) ( Figure S3B ) was also upregulated in CML CD34 + cells. Since the validation of targets obtained from gene expression data can pose a substantial challenge, mainly due to the low abundance of CD34 + cells in both CML and mPB donors, we utilized NanoString technology, which can generate high-quality gene expression results even from 100 ng of RNA. Based on Bloodspot ( https://servers.binf.ku.dk/bloodspot/ ) and previous studies ( 8 , 11 , 16 ), a 21-gene panel of potentially differentially expressed genes was custom-designed. The expression of these 21 genes was evaluated in an independent cohort of CML-CD34 + (n = 39) and mPB-CD34 + cells (n = 9). We observed significant upregulation of CTSG and ELANE (Fig. 3A) and considerable downregulation of CDH2, S100A8, S100A9 , and RXRA in CML CD34 + cells compared to the mPB CD34 + (Fig. 3B), in line with the transcriptomic results. The result was further corroborated by an immunoblotting experiment showing increased CTSG protein expression in CML CD34 + cells compared to mPB CD34 + cells (Fig. 3F). Further, we tested the expression of CTSG in CML primary samples using q-RT-PCR. There was significant upregulation of CTSG in treatment-naïve CML patients compared to healthy donor granulocytes, as depicted in Fig. 3C . However, no significant difference in CTSG expression was observed between CML patients who achieved major molecular response (MMR) and those who did not (Fig. 3D), suggesting that CTSG expression may not directly correlate with response to TKI therapy or disease prognosis in CML. Further, we tested CTSG expression in CML patients presenting at blast crisis (BC) compared to those in the chronic phase (CP). CTSG expression was markedly reduced in CML blast crisis patients (Fig. 3E), since CTSG is mainly present in the azurophilic granules in the myeloid compartment and hence has low expression in blasts. Molecular inhibition of CTSG reduces colony-forming capacity and leukemogenic potential in vivo : To evaluate the functional significance of CTSG in CML, we carried out CTSG knockdown ( CTSG KD) in the CML cell line. The experimental design is outlined in Fig. 4A. Our initial screening of CML cell lines revealed that the EM-2 cell line expressed the highest level of CTSG protein expression (Fig. 4B). Hence, we transduced EM-2 cells with doxycycline (dox)-inducible CTSG shRNAs ( CTSG KD). After five days of dox induction, CTSG KD cells showed a marked reduction in the CTSG protein expression compared with the scramble, confirming knockdown (Fig. 4C). We assessed the effect of knockdown in the proliferation and colony-forming capacity. EM-2 CTSG KD cells did not show any difference in proliferation ( Figure S4A ) but exhibited a reduced colony-forming capacity, supporting the possible role of CTSG in the maintenance of CML cell stemness (Fig. 4D). To investigate the impact of CTSG suppression of self-renewal potential in-vivo , we transplanted EM-2 CTSG KD cells, with and without doxycycline induction, into sublethally irradiated NSG mice (Fig. 4E).CTSG knockdown significantly reduced the repopulating potential leading to increased overall survival of the mice compared to the control group (Fig. 4F-G). These findings suggest that CTSG may be a promising target for therapeutic intervention in CML. Testing pharmacological inhibition of CTSG Jin et al. demonstrated that Ponasterone A treatment reduces CTSG protein expression in an AML cell line. Based on this finding, we investigated the effects of Ponasterone A on CML CD34 + cells and mPB CD34 + ( 15 ). Treatment with Ponasterone A consistently led to a dose-dependent decrease in CTSG protein expression (Fig. 5A) and a subsequent reduction in colony-forming capacity in CML CD34 + cells (Fig. 5B). In contrast, Ponasterone A treatment did not have any effect on the colony-forming capacity of mPB CD34 + ( Figure S4B ). These results suggest that pharmacological inhibition of CTSG may reduce the colony-forming capacity of CML CD34 + cells, indicating a potential role for CTSG in the self-renewal of CML cells. Further investigations are warranted to elucidate the precise mechanisms by which CTSG influences leukemogenesis and to determine the specific downstream pathways or cellular processes affected by its suppression. Understanding the molecular and cellular consequences of CTSG knockdown in the context of CML will provide valuable insights for developing targeted therapies to disrupt the leukemogenic potential of CML cells. Discussion Chronic myeloid leukemia (CML) is driven by leukemic stem/progenitor cells (LSPCs) that sustain disease progression and resistance to therapy. Understanding the molecular signature of these cells is critical for developing targeted treatment. Transcriptomic profiling of CML CD34 + cells has revealed notable alterations in gene expression, with 1,563 genes upregulated and 1,400 genes downregulated. Pathway enrichment analysis has pinpointed significant changes in genes associated with the cell cycle, the Fanconi anemia pathway, and metabolism in CML CD34 + cells, indicating a shift towards enhanced oxidative phosphorylation (OxPHOS), akin to previous findings (9). Upregulation of genes such as FANCI, FANCM , FANCB , FANCG , and FANCD2 within the Fanconi anemia pathway suggests potential enhancement of DNA repair mechanisms. Similarly, metabolic genes related to ETC-complex I and fatty acid β oxidation exhibited significant upregulation, indicating altered metabolism in CML CD34+ cells. These findings underscore the dysregulated metabolism and enhanced DNA repair mechanisms characteristic of CML CD34 + cells. The dysregulation of the Fanconi anemia pathway has been previously implicated in CML CD34 + cells, leading to chromosome instability (17). Additionally, altered mitochondrial metabolism, as evidenced by the upregulation of ETC-I genes, can exacerbate oxidative stress and DNA damage, further contributing to genomic instability (18). Furthermore, changes in cell surface marker expression have been observed in CML CD34 + cells, with upregulation of CD200R1, CD276, CD96, and CD36 and downregulation of CD79A , CD37 and CD22 . Notably, CD36 upregulation in CD34+CD38low CML cells has been linked to reduced responsiveness to imatinib, yet increased vulnerability to antibody-based therapies (19). CD96, a recognized LSC marker in AML, and CD276, an immune checkpoint inhibitor, a marker highly expressed in monocytic AML, are therapeutically relevant targets. Preclinical studies have shown promising results in the development of CAR-T therapies targeting CD276 (20–22). The downregulation of CD79A , CD37 , and CD22 suggests perturbed B-cell signaling and activation in CML, contributing to the selective accumulation of myeloid cells and a reduction in the lymphoid lineage. While previous studies have reported elevated expression of CD26 and CD25 in CML LSCs (23,24), no significant differences were observed in our study, possibly due to the use of CML CD34 + cells rather than the more primitive CML CD34 + CD38 - subset. Moreover, our results demonstrate significant upregulation of serine proteases in CML CD34 + cells, implicating their potential role in LSC survival. Serine proteases, including proteinase 3 (P3) and neutrophil elastase (ELANE), have been implicated in cancer development and progression, with potential immunomodulatory effects (25). Further validation confirmed the upregulation of CTSG and ELANE, primary granule proteins, and the downregulation of S100A8/A9, secondary granule proteins. CTSG has been associated with immune response regulation and may serve as a potential therapeutic target in CML (24). In addition, Molecular inhibition of CTSG impaired self-renewal properties in a CDX mouse model in vivo. Pharmacological inhibition of CTSG showed a dose-dependent reduction of CTSG and reduced colony-forming capacity in primary CD34 + CML cells. These results suggest the potential role of CTSG in the maintenance of CML LSPCs. We compared our datasets with publicly available datasets to identify the LSPC-specific gene signature. This comparative approach reduces dataset-specific biases and highlights robust, reproducible changes in gene expression. Genes involved in heme metabolism, porphyrin metabolism, and heme biosynthesis were consistently upregulated in CML CD34 + cells. These pathways are essential for iron handling and redox biology, which are critical for cell proliferation and survival. Genes related to cellular and leukocyte homeostasis were commonly downregulated, suggesting impaired normal hematopoietic regulation in leukemic progenitors. The identification of 28 upregulated and 18 downregulated genes across datasets forms a robust molecular signature of CML LSPCs. This signature implicates dysregulated iron metabolism as a hallmark of these cells. Iron and heme metabolism are tightly linked to cellular energy production, oxidative stress, and DNA synthesis. Dysregulation of these pathways may provide CML LSPCs with survival advantages or contribute to therapy resistance. Targeting iron metabolism could selectively affect leukemic stem cells while sparing normal hematopoietic cells. In summary, Global transcriptomic analysis of CML vs. normal CD34 + cells provided valuable insights into the molecular intricacies of CML, highlighting altered pathways, cell surface markers, and the potential role of CTSG in LSPC survival. These findings offer a foundation for future research to develop targeted therapeutic strategies to disrupt these survival mechanisms and improve treatment outcomes for CML patients. Declarations Author Contribution ESB, SI, BMR, AM, and MRA contributed to setting up all the in-vitro and in-vivo experiments, and transcriptome experiments, including analysis. AA and VM provided primary patient material and clinical response results of the patients. SRV provided input on data analysis and troubleshooting in-vitro experiments.PB procured research funding, designed the study, analyzed data, and provided a critical review of the manuscript. All the authors reviewed the manuscript, suggested comments, and approved the final version of the manuscript. Acknowledgement Technical assistance provided by Ms. Sangeetha is gratefully acknowledged. We thank the staff of the flow cytometry facility of the Centre for Stem Cell Research, Bagayam, Vellore, for their help. Data Availability statement: The datasets generated and/or analysed during the current study are available in the gene expression omnibus (GEO) repository, GSE315681. Conflict-of-interest disclosure The authors declare no conflict of interest to disclose. References Chu, S. et al. Persistence of leukemia stem cells in chronic myelogenous leukemia patients in prolonged remission with imatinib treatment. Blood 118 (20), 5565–5572 (2011). Corbin, A. S. et al. Human chronic myeloid leukemia stem cells are insensitive to imatinib despite inhibition of BCR-ABL activity. J. Clin. Invest. 121 (1), 396–409 (2011). Kuepper, M. K. et al. Stem cell persistence in CML is mediated by extrinsically activated JAK1-STAT3 signaling. Leukemia 33 (8), 1964–1977 (2019). Wang, W. Z. et al. Silencing of miR-21 sensitizes CML CD34 + stem/progenitor cells to imatinib-induced apoptosis by blocking PI3K/AKT pathway. Leuk. Res. 39 (10), 1117–1124 (2015). Dierks, C. et al. 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Hematol. 51 , 17–24 (2017). Khan, M. et al. Cathepsin G Is Expressed by Acute Lymphoblastic Leukemia and Is a Potential Immunotherapeutic Target. Front. Immunol. 8 , 1975 (2017). Additional Declarations No competing interests reported. Supplementary Files Slide6.jpg Supplementary Figure 1: Schematic representation of the work outline of global transcriptomics. Slide7.jpg Supplementary Figure 2. Pathways dysregulated in significantly upregulated and downregulated genes: Enrichment analysis was performed using Enrichr A) Reactome 2021 B) GO cellular components and C) GO molecular functions. Pathways dysregulated in significantly downregulated genes: Enrichment analysis was performed using Enrichr D) Reactome 2021 D E) Go cellular components and F) GO molecular functions of significantly enriched pathways were manually curated based on the smallest p-values obtained across all the databases. Slide8.jpg Supplementary Figure 3A) Altered Cell surface markers in CML CD34 + cells compared with mPB CD34+ cells. B) Aberrant expression of serine proteases in CML CD34 + cells. The Y-axis is in the log 10 scale. Slide9.jpg Supplementary Figure 4A) Effect of CTSG KD in the proliferation in EM-2 cell line B) Effect of Ponasterone A in mobilized peripheral blood CD34 + cells. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8580579","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":584745600,"identity":"f5ab4a92-0162-4726-838b-7aeb4d8ac1a5","order_by":0,"name":"Esther Sathya Bama Benjamin","email":"","orcid":"","institution":"Christian Medical College","correspondingAuthor":false,"prefix":"","firstName":"Esther","middleName":"Sathya Bama","lastName":"Benjamin","suffix":""},{"id":584745601,"identity":"e08b9fbf-3aae-41e3-9293-8ecb73b75d73","order_by":1,"name":"Raveen Stephen Stallon Illangeswaran","email":"","orcid":"","institution":"Christian Medical College","correspondingAuthor":false,"prefix":"","firstName":"Raveen","middleName":"Stephen Stallon","lastName":"Illangeswaran","suffix":""},{"id":584745604,"identity":"58506a69-59f6-4b8d-8e40-d7bcb496be0a","order_by":2,"name":"Bharathi M Rajamani","email":"","orcid":"","institution":"Christian Medical College","correspondingAuthor":false,"prefix":"","firstName":"Bharathi","middleName":"M","lastName":"Rajamani","suffix":""},{"id":584745605,"identity":"524fadf2-593c-43f3-872e-9f164e5cb320","order_by":3,"name":"Ajith Mohan","email":"","orcid":"","institution":"Christian Medical College","correspondingAuthor":false,"prefix":"","firstName":"Ajith","middleName":"","lastName":"Mohan","suffix":""},{"id":584745607,"identity":"b027f60b-4896-4195-b262-803223061257","order_by":4,"name":"Manasi Arun Rane","email":"","orcid":"","institution":"Christian Medical College","correspondingAuthor":false,"prefix":"","firstName":"Manasi","middleName":"Arun","lastName":"Rane","suffix":""},{"id":584745608,"identity":"332d62bc-a0e1-4c54-8a93-50ad5b328f0e","order_by":5,"name":"Aby Abraham","email":"","orcid":"","institution":"Christian Medical College","correspondingAuthor":false,"prefix":"","firstName":"Aby","middleName":"","lastName":"Abraham","suffix":""},{"id":584745610,"identity":"8f9f188a-419c-4408-b86d-2893e25814fc","order_by":6,"name":"Vikram Mathews","email":"","orcid":"","institution":"Christian Medical College","correspondingAuthor":false,"prefix":"","firstName":"Vikram","middleName":"","lastName":"Mathews","suffix":""},{"id":584745612,"identity":"3a202012-e66c-41f9-831e-f2d06f01d699","order_by":7,"name":"Shaji R Velayudhan","email":"","orcid":"","institution":"Christian Medical College","correspondingAuthor":false,"prefix":"","firstName":"Shaji","middleName":"R","lastName":"Velayudhan","suffix":""},{"id":584745613,"identity":"736c576b-d93f-4a9b-be75-3f85cc9f562c","order_by":8,"name":"Poonkuzhali Balasubramanian","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFUlEQVRIie2RsWrDMBBATxiSRZDVJRD/gkKhwTQ0v5JgaJcU8gEZZDpkCXT1UOgvdOrU4cSBsxiyCrzUFDq7dOnWSoIsrRw6ZtAbxJ2Od9xJAIHACSLciTaIJLYui3I8lPG4wqQqXMbk/5WI/1Z8TIZ36m1V0WzSVzlN18+J2OUSP16mMNggo9VfJX0os/NCU5RuF5KWZT1+qpSZ8P0a4moOVHgG08uLIW+pJ9Aot7KenxUm4EgAGoB4t8LFvpGUWuWxsco3JEcVTbHQpjkzyiBmVkFT8ituF17dCKEbqbZml3u+MLtgxseVnbDjxXh5ORP7jNqvdZ30+qReW7wajXZEnx4FYs+dg9sP8lY6lUAgEAgc+AFq7HlpYiX2gwAAAABJRU5ErkJggg==","orcid":"","institution":"Christian Medical College","correspondingAuthor":true,"prefix":"","firstName":"Poonkuzhali","middleName":"","lastName":"Balasubramanian","suffix":""}],"badges":[],"createdAt":"2026-01-12 10:38:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8580579/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8580579/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101881612,"identity":"64972235-bdc2-485f-aa3a-02693192ae2c","added_by":"auto","created_at":"2026-02-04 15:13:56","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":156001,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlobal transcriptomics analysis of CML and mPB CD34\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e cells.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Principal component analysis (PCA) plot of CML CD34\u003csup\u003e+\u003c/sup\u003e and mPB CD34\u003csup\u003e+\u003c/sup\u003e cells. (\u003cstrong\u003eB\u003c/strong\u003e) Volcano plot showing the significant up and downregulated genes in the CML CD34\u003csup\u003e+\u003c/sup\u003e cells. Red dots indicate upregulated genes and Blue dots indicate downregulated genes between CML and mPB CD34\u003csup\u003e+\u003c/sup\u003e cells (\u003cstrong\u003eC\u003c/strong\u003e) Heatmap visualizing the expression of the significantly differentially expressed genes showing top-up and downregulated genes with a 2-fold difference (FDR-adjusted p \u0026lt; 0.05) between CML and normal CD34\u003csup\u003e+\u003c/sup\u003e. Pathways dysregulated in significantly upregulated genes using Enrichr \u003cstrong\u003eD\u003c/strong\u003e) MsigDB Hallmark 2020 \u003cstrong\u003eE\u003c/strong\u003e) KEGG 2021 \u003cstrong\u003eF\u003c/strong\u003e) GO Biological Processors and downregulated genes \u003cstrong\u003eG\u003c/strong\u003e) MsigDB Hallmark 2020 \u003cstrong\u003eH\u003c/strong\u003e) KEGG 2021 \u003cstrong\u003eI\u003c/strong\u003e) GO Biological Processors of significantly enriched pathways were manually curated based on the smallest p-values obtained across all the databases.\u003c/p\u003e","description":"","filename":"Slide1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8580579/v1/8686773935b797afe84b1e8b.jpg"},{"id":101881615,"identity":"ab7e2272-3093-4051-a533-1e050dea026d","added_by":"auto","created_at":"2026-02-04 15:14:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":105891,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene signature of CML CD34\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e cell. A) \u003c/strong\u003eGO biological process\u003cstrong\u003e B) \u003c/strong\u003eReactome analysis of commonly upregulated genes in CML CD34\u003csup\u003e+ \u003c/sup\u003ecells\u003cstrong\u003e C) \u003c/strong\u003eGO biological process of commonly downregulated genes in CML CD34\u003csup\u003e+\u003c/sup\u003e cells \u003cstrong\u003eD) \u003c/strong\u003eVenn Diagram showing number of genes differentially expressed in CML CD34\u003csup\u003e+\u003c/sup\u003e compared with other datasets\u003c/p\u003e","description":"","filename":"Slide2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8580579/v1/16576ff70b90e7061e8a91e5.jpg"},{"id":101867752,"identity":"66d09392-7628-45c6-b7a7-a8b031d6d9fc","added_by":"auto","created_at":"2026-02-04 12:43:47","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":90553,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of differentially expressed genes in CML vs. normal CD34\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003ecells.\u003c/strong\u003e Bar graph showing log fold change of selected target genes in CML CD34\u003csup\u003e+\u003c/sup\u003e \u0026nbsp;and mPB CD34\u003csup\u003e+\u003c/sup\u003e cells normalized with housekeeping gene \u003cstrong\u003eA\u003c/strong\u003e) Upregulated \u003cstrong\u003eB\u003c/strong\u003e) Downregulated. The target gene expression was normalized with the housekeeping genes (\u003cem\u003eGUSB\u003c/em\u003e, \u003cem\u003eGAPDH\u003c/em\u003e, and \u003cem\u003eACTB\u003c/em\u003e). qRT-PCR analysis of transcript levels of \u003cem\u003eCTSG\u003c/em\u003e in \u003cstrong\u003eC\u003c/strong\u003e) Treatment naïve CP- CML (n = 12) vs. Healthy donor (HD) granulocytes (n = 5) \u003cstrong\u003eD\u003c/strong\u003e) CML patients who achieved MMR (n=6) vs. no MMR with TKI (n = 7) \u003cstrong\u003eE\u003c/strong\u003e) CML patients at chronic phase(CP)(n=11), accelerated phase (AP)(n=8) and Blast crisis (BC)(n=5) normalized with \u003cem\u003eACTB\u003c/em\u003e (housekeeping gene)\u003cstrong\u003e F\u003c/strong\u003e) Western blot analysis of CTSG levels in CML CD34\u003csup\u003e+\u003c/sup\u003e(n=2) and normal CD34\u003csup\u003e+\u003c/sup\u003e(n=2). Β-actin was used as the loading control.\u003cstrong\u003e The \u003c/strong\u003eY-axis is in the log\u003csub\u003e10 \u003c/sub\u003escale for qRT-PCR analysis\u003c/p\u003e","description":"","filename":"Slide3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8580579/v1/014f9690f62f3ea0ea0efde0.jpg"},{"id":101881656,"identity":"f4f22c4c-3f50-4d0e-ad78-79c52c5646fa","added_by":"auto","created_at":"2026-02-04 15:14:31","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":81976,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCTSG knockdown reduces stemness and leukemogenic potential: A) \u003c/strong\u003eExperimental outline of CTSG KD in EM-2 cell line; \u003cstrong\u003eB) \u003c/strong\u003eWestern blot analysis of CTSG protein expression across CML cell lines \u003cstrong\u003eC) \u003c/strong\u003eCTSG\u003cstrong\u003e \u003c/strong\u003eprotein expression before and after KD in EM2 cells. β-actin was used as the loading control. \u003cstrong\u003eD) \u003c/strong\u003eColony formation assay in EM-2 CTSG KD and scramble.\u003cstrong\u003eE\u003c/strong\u003e) Work outline of a Cell line-derived xenograft model. CTSG KD cells were injected into sublethally irradiated (200cGy) NSG mice. After transplantation, five mice (n=5) were supplemented with doxycycline (Dox+), and their survival was compared to a Dox-negative (Dox-) group (n=5). \u003cstrong\u003eF)\u003c/strong\u003e Representative in vivo bioluminescence imaging showing luciferase expression in both Dox+ and Dox− groups. \u003cstrong\u003eG)\u003c/strong\u003e Kaplan–Meier survival curve of NSG mice transplanted with CTSG KD cells, comparing survival between Dox+ and Dox-groups.\u003c/p\u003e","description":"","filename":"Slide4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8580579/v1/35101cd4785c206b29685cc0.jpg"},{"id":101867759,"identity":"d5966f0e-9a26-42c8-86e1-f15a3e19d455","added_by":"auto","created_at":"2026-02-04 12:43:48","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":47310,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePonasterone treatment reduced the colony-forming capacity of CML CD34\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e cells\u003c/strong\u003e. CML CD34+ cells treated with Ponasterone A at different concentrations showed dose-dependent reduction of A) CTSG protein expression and B) colony-forming capacity.\u003c/p\u003e","description":"","filename":"Slide5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8580579/v1/73c0666bcabe311c75e16830.jpg"},{"id":101883448,"identity":"5ea54606-9560-402c-85bb-ce84b368ffbf","added_by":"auto","created_at":"2026-02-04 15:28:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1595604,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8580579/v1/5b4e5c66-6a2e-4e3c-8c1e-e9f374543bf3.pdf"},{"id":101881904,"identity":"a9acb714-7f0f-438f-b837-28eda5d4b034","added_by":"auto","created_at":"2026-02-04 15:17:32","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":32835,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1: Schematic representation of the work outline of global transcriptomics.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Slide6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8580579/v1/c9b119e53b9d9dd025bf5adc.jpg"},{"id":101881845,"identity":"08a28ed5-a132-41c1-826c-7e0ec7343a38","added_by":"auto","created_at":"2026-02-04 15:17:03","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":100437,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2. Pathways dysregulated in significantly upregulated and downregulated genes:\u003c/strong\u003e Enrichment analysis was performed using Enrichr \u003cstrong\u003eA\u003c/strong\u003e) Reactome 2021 \u003cstrong\u003eB\u003c/strong\u003e) GO cellular components and \u003cstrong\u003eC\u003c/strong\u003e) GO molecular functions. Pathways dysregulated in significantly downregulated genes: Enrichment analysis was performed using Enrichr \u003cstrong\u003eD\u003c/strong\u003e) Reactome 2021 D \u003cstrong\u003eE\u003c/strong\u003e) Go cellular components and \u003cstrong\u003eF\u003c/strong\u003e) GO molecular functions of significantly enriched pathways were manually curated based on the smallest p-values obtained across all the databases.\u003c/p\u003e","description":"","filename":"Slide7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8580579/v1/67cbdbed96eb391db13d7920.jpg"},{"id":101881510,"identity":"28285811-f5bf-43e8-8f43-d438732d8acd","added_by":"auto","created_at":"2026-02-04 15:12:37","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":106402,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3A\u003c/strong\u003e) Altered Cell surface markers in CML CD34\u003csup\u003e+\u003c/sup\u003e cells compared with mPB CD34+ cells.\u0026nbsp; \u003cstrong\u003eB)\u003c/strong\u003e Aberrant expression of serine proteases in CML CD34\u003csup\u003e+\u003c/sup\u003e cells. The Y-axis is in the log\u003csub\u003e10 \u003c/sub\u003escale.\u003c/p\u003e","description":"","filename":"Slide8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8580579/v1/74e730d79973edbf9918f783.jpg"},{"id":101881707,"identity":"2a51468b-fa79-4dea-ad77-d684b5e47db6","added_by":"auto","created_at":"2026-02-04 15:15:03","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":45045,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 4A) \u003c/strong\u003eEffect of CTSG KD in the proliferation in EM-2 cell line \u003cstrong\u003eB) \u003c/strong\u003eEffect of Ponasterone A in mobilized peripheral blood CD34\u003csup\u003e+ \u003c/sup\u003ecells\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Slide9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8580579/v1/d034fbc09ea66a928aef91b1.jpg"},{"id":101867757,"identity":"7f843efc-0736-41de-9a7b-6af4a14ff6d5","added_by":"auto","created_at":"2026-02-04 12:43:47","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":68531,"visible":true,"origin":"","legend":"","description":"","filename":"SupplTable1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8580579/v1/edfcbd08ffa63c80c6d77be6.jpg"},{"id":101881708,"identity":"1a0803d0-947c-44fe-a6c7-4799b2860eac","added_by":"auto","created_at":"2026-02-04 15:15:04","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":58989,"visible":true,"origin":"","legend":"","description":"","filename":"SupplTable2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8580579/v1/310fb377253f4862737acc29.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Targeting Cathepsin-G to Overcome Leukemic Stem Cell Persistence in Chronic Myeloid","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTyrosine kinase inhibitors (TKIs) have revolutionized the management of chronic myeloid leukemia (CML) by significantly improving treatment outcomes. However, resistance to TKIs remains a challenge in some patients. While mutations in the BCR-ABL1 kinase domain explain resistance in certain cases, the cause of resistance remains unclear in the majority of patients. Emerging evidence suggests that leukemic stem cells (LSCs) may play a critical role in therapy resistance by evading TKI-mediated targeting through \u003cem\u003eBCR-ABL1\u003c/em\u003e-independent mechanisms. Unlike bulk leukemic cells, LSCs exhibit intrinsic resistance to TKIs (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Understanding the molecular mechanisms underlying LSC resistance could aid in the development of targeted therapeutic strategies to selectively eliminate these cells. One approach involves comparing transcriptomic profiles between healthy and CML stem cells to identify specific gene signatures unique to CML stem cells. In the absence of definitive cell surface markers distinguishing CML LSCs from normal hematopoietic stem cells (HSCs), CD34\u0026thinsp;+\u0026thinsp;cells are often considered a marker for leukemic stem and progenitor cells (LSPCs) in many studies.\u003c/p\u003e \u003cp\u003eDespite efforts to characterize differences between CML cells and normal HSCs, only a few studies have employed CML CD34\u0026thinsp;+\u0026thinsp;cells to identify these distinct signatures (\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Interestingly, the metabolic profiling of CML CD34\u003csup\u003e+\u003c/sup\u003e cells has revealed distinct metabolic differences, highlighting enhanced oxidative phosphorylation (OxPHOS) when compared to healthy HSCs (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Targeting these metabolic vulnerabilities has shown promise in preclinical studies. For instance, tigecycline, an antibiotic, also effectively inhibited OxPHOS in treatment-resistant CML LSCs, both in vitro and in vivo, while sparing normal HSCs. However, in a Phase I trial involving patients with relapsed/refractory AML, tigecycline demonstrated a satisfactory safety profile but failed to yield significant clinical benefits, likely due to its shorter half-life (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). These findings underscore the need to explore alternative strategies for targeting LSCs.\u003c/p\u003e \u003cp\u003ePrevious reports on transcriptomic signatures in CML have highlighted an increased expression of focal adhesion-related genes in CML CD34\u003csup\u003e+\u003c/sup\u003e cells and identified Integrin-linked kinase (ILK) as a crucial focal adhesion component required for LSC self-renewal in vivo. The study predominantly utilized in vitro and in vivo models to assess the effects of ILK inhibition (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Similarly, Zhao et al. (2022) reported the role of \u003cem\u003eMS4A3\u003c/em\u003e (Membrane spanning 4-domains A3) as a functional tumor suppressor in CML, where its downregulation resulted in the retention of leukemia stem/progenitor cells (LSPCs) in a state of differentiation blockage that rendered them insensitive to TKIs. In this recent study, exogenous MS4A3 protein was introduced using a targeted delivery strategy, although the feasibility of clinical translation for this approach remains uncertain (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Overall, previous studies highlight the significance of considering metabolic differences and alternative signaling pathways in unraveling treatment-resistant cells.\u003c/p\u003e \u003cp\u003eIn the present study, using a global transcriptomics approach, we aimed to identify gene signatures and pathways differentially expressed in CML CD34\u003csup\u003e+\u003c/sup\u003e cells to better understand the mechanisms contributing to CML LSC survival.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics and IRB statement\u003c/h2\u003e \u003cp\u003e \u003cb\u003eAll the study procedures and methods were performed in accordance with the Declaration of Helsinki and Institutional guidelines.\u003c/b\u003e The experimental protocols were reviewed and approved by institutional review Board (IRB), Christian Medical College, Vellore (IRB Min No. 14754 dated 27.07.2022). Informed Consent was obtained from all the participants prior to inclusion in this study.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePrimary cells and Cell lines\u003c/h3\u003e\n\u003cp\u003eAfter obtaining written informed consent, Peripheral blood / Bone marrow (BM) aspirates were collected from patients with chronic phase CML at diagnosis. The experimental protocols were reviewed and approved by institutional review Board (IRB), Christian Medical College, Vellore (IRB Min No. 14754 dated 27.07.2022). Informed Consent was obtained from all the participants prior to inclusion in this study. EM-2, KU812, Lama84, KCL22, KYO, MEG01, JURL-MK1, and K562 cell lines were procured from DSMZ, German Collection of Microorganisms and Cell Cultures. All the cell lines were maintained in RPMI supplemented with 10% Fetal Bovine Serum (Gibco) and 1% Penicillin and streptomycin (Gibco). All cell lines were periodically tested for mycoplasma (Universal Mycoplasma Detection Kit, ATCC) and were mycoplasma-free.\u003c/p\u003e\n\u003ch3\u003eIsolation and culture of CD34 + cells\u003c/h3\u003e\n\u003cp\u003eBriefly, Mononuclear cells (MNCs) were isolated using Lymphoprep (STEMCELL Technologies) by density gradient separation, and CD34\u003csup\u003e+\u003c/sup\u003e cells were enriched immunomagnetically using an EasySep CD34 Positive Selection Kit (STEMCELL Technologies) following the manufacturer's protocol. Purity was verified by re-staining isolated cells with an allophycocyanin-labeled (APC) anti-CD34 antibody (Biolegend) and analyzing cells on a FACS Accuri C6 (BD) instrument. Cryopreserved CD34\u003csup\u003e+\u003c/sup\u003e cells of the CML patients and the normal donor were thawed and cultured in StemSpan\u0026trade; SFEM II medium (Stem Cell Technologies) supplemented with CD34\u003csup\u003e+\u003c/sup\u003e Expansion Supplement (Stem Cell Technologies) and UM729 (Stem Cell Technologies). The cells were cultured for 6\u0026ndash;7 days with half-medium change every other day to assess the cell proliferation.\u003c/p\u003e\n\u003ch3\u003ecDNA synthesis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from PB/BM Cells using TRI reagent (Sigma, St. Louis, USA) per the manufacturer's protocol. Following the manufacturer's instructions, 2\u0026micro;g of total RNA was converted to cDNA using a High-Capacity Reverse Transcription Kit (ThermoFisher Scientific) and stored at -20\u0026deg;C for further downstream applications. Confirmation of cDNA conversion was done using RT-PCR to test the expression of housekeeping gene β-glucuronidase (\u003cem\u003eGUS\u003c/em\u003e) (Table\u0026nbsp;3.2).\u003c/p\u003e\n\u003ch3\u003eStrand-specific RNA-sequencing\u003c/h3\u003e\n\u003cp\u003eRNA was extracted from CML CD34\u003csup\u003e+\u003c/sup\u003e (N\u0026thinsp;=\u0026thinsp;10) and mobilized peripheral blood (mPB) CD34\u003csup\u003e+\u003c/sup\u003e (N\u0026thinsp;=\u0026thinsp;4) cells using Total RNA Purification Plus Kit (Norgen Biotek) as described earlier and then subjected to Strand-specific transcriptomics. RNA QC was evaluated using Agilent Bioanalyzer 2100 and Qubit High sensitivity RNA assay. The transcriptome profile of CML CD34\u003csup\u003e+\u003c/sup\u003e cells was compared to the mPB CD34\u003csup\u003e+\u003c/sup\u003e cells. The raw reads were filtered using Trimmomatic for quality scores and adapters. The filtered reads were aligned to the Human genome (hg19) using splice-aware aligner like HISAT2 to quantify reads mapped to each transcript. The alignment percentage of the reads was in the range of 5.5\u0026ndash;97.5% for all the samples. The total number of uniquely mapped reads was counted using feature counts. The uniquely mapped reads were then subjected to differential gene expression using Deseq2 (Figure S1). The datasets generated and/or analysed during the current study are available in the gene expression omnibus (GEO) repository, GSE315681.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNanoString nCounter Plexset custom panel\u003c/h2\u003e \u003cp\u003eBased on the differentially expressed genes list obtained from the transcriptomic data, we custom-designed a gene panel consisting of 21 target genes associated with cell surface markers, transporters, cell adhesion, and immune regulation based on previous publications (Supplementary Table\u0026nbsp;2). The expression of these 21 genes was then examined in an independent cohort of CML CD34\u003csup\u003e+\u003c/sup\u003e (N\u0026thinsp;=\u0026thinsp;39) and mPB CD34\u003csup\u003e+\u003c/sup\u003e (N\u0026thinsp;=\u0026thinsp;9) cells. Read counts were normalized using positive control and three housekeeping genes (\u003cem\u003eACTB\u003c/em\u003e, \u003cem\u003eGAPDH\u003c/em\u003e, and \u003cem\u003eGUSB\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunoblot\u003c/h3\u003e\n\u003cp\u003eProtein lysates were prepared using radioimmunoprecipitation assay buffer supplemented with a protease inhibitor mixture and phenylmethylsulfonylfluoride. 30 \u0026micro;g of the cell lysate was loaded onto a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with non-fat dry milk powder in tris-buffered saline containing Tween 20 and incubated with primary antibodies for CTSG and β-actin. The proteins were detected using the ECL chemiluminescence kit and imaged using the FluorChem E system (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eLentiviral-mediated\u003c/b\u003e \u003cb\u003eCTSG\u003c/b\u003e \u003cb\u003eknockdown in CML cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEM-2 cells were seeded at 2x10\u003csup\u003e5\u003c/sup\u003e cells/24-well and infected with 20ul of the concentrated lentiviral vector containing Doxycyline inducible shRNA constructs targeting human CTSG or a non-targeting sequence (Tet-pLKO-puro (Addgene: 21915)) in the presence of polybrene for 24 hours. The expression of the shRNA was induced with doxycycline (Dox) (1\u0026micro;g/ml) for 5 days; subsequently, western blotting was performed to confirm the knockdown (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eColony-forming unit assay\u003c/h3\u003e\n\u003cp\u003eBriefly, 1,000 cells were mixed in 1 mL MethoCult (STEMCELL Technologies), and then the number of colonies was scored on day 14 following the manufacturer's protocol.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell line-derived Xenograft model\u003c/h2\u003e \u003cp\u003eEM-2 cells were transduced with lentivirus containing the Dox inducible shCTSG, followed by Dox induction for 48h. These cells induced with and without dox were transplanted into sub-lethally irradiated (2 Gy) 8\u0026ndash;10 weeks old NSG mice (N\u0026thinsp;=\u0026thinsp;9 each) (The Jackson Laboratory, Bar Harbor, ME, USA) via the tail vein. Doxycycline was provided in drinking water at a concentration of 1mg/ml. The median survival of mice was evaluated. The difference in percentage survival between the CTSG WT and KD mice was evaluated by Kaplan-Meier survival analysis, and the median survival was compared using the Mann-Whitney U test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eTranscriptome profiling in primary CML CD34\u003c/b\u003e \u003csup\u003e \u003cb\u003e+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003ecells reveals unique gene signatures compared to normal CD34\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003ecells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo identify the unique gene signature of CML LSPCs, we conducted transcriptomic profiling of CML CD34\u0026thinsp;+\u0026thinsp;and mPB CD34\u0026thinsp;+\u0026thinsp;cells using NGS on an Illumina sequencing platform. Global transcriptome profiling of differentially expressed genes was evaluated by filtering based on the reads per kilobase million (RPKM)\u0026thinsp;\u0026gt;\u0026thinsp;1 and a minimum of a 2-fold difference (CuffDiff, FDR-adjusted p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between CML CD34\u003csup\u003e+\u003c/sup\u003e and mPB CD34\u003csup\u003e+\u003c/sup\u003e cells. Principal Component Analysis (PCA) revealed a distinct separation between the two groups, indicating an apparent clustering of samples that aligned with their respective groups. (Fig.\u0026nbsp;1A). We observed 1563 genes upregulated and 1400 genes downregulated in CML CD34\u003csup\u003e+\u003c/sup\u003e cells compared to mPB CD34\u003csup\u003e+\u003c/sup\u003e (Fig.\u0026nbsp;1B). Top upregulated genes include \u003cem\u003eGSTM1\u003c/em\u003e (Glutathione S-transferase mu 1), \u003cem\u003eAZU1\u003c/em\u003e (Azurocidin 1), \u003cem\u003eLTF\u003c/em\u003e (Lactotransferrin\u003cb\u003e)\u003c/b\u003e, \u003cem\u003eCTSG\u003c/em\u003e (Cathepsin G), \u003cem\u003eHIST14C\u003c/em\u003e (histone proteins) and downregulated genes include \u003cem\u003eCXCL13\u003c/em\u003e (C-X-C ligand 13), \u003cem\u003eITGA3\u003c/em\u003e (Integrin Subunit Alpha 3), \u003cem\u003eCDH4\u003c/em\u003e (Cadherin 4), \u003cem\u003ePRMD16\u003c/em\u003e (protein arginine methyltransferase 16) (Fig.\u0026nbsp;1C). Pathway enrichment analysis revealed that the upregulated genes are involved in immune response, cytokine signaling, and cell adhesion. Pathway enrichment analysis of the top differentially expressed genes (log\u003csub\u003e2\u003c/sub\u003eF\u0026thinsp;\u0026gt;\u0026thinsp;1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was done using Enrichr (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://maayanlab.cloud/Enrichr/\u003c/span\u003e\u003cspan address=\"https://maayanlab.cloud/Enrichr/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Based on Reactome, KEGG, and MSigDB datasets, genes associated with the cell cycle, Fanconi anemia pathway, and metabolism were significantly upregulated in CML CD34\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;1D-E \u003cb\u003eand S2A\u003c/b\u003e). Gene ontology analysis using GO biological process, GO cellular component, and GO molecular function revealed that genes associated with mitotic sister chromatid segregation spindle, single-strand helicase activity, and mitochondrial matrix were dysregulated in CML CD34\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;1F \u003cb\u003eand S2B-C\u003c/b\u003e).\u003c/p\u003e \u003cp\u003ePathway enrichment of significantly downregulated genes revealed the genes associated with cytokine signaling, inflammatory response, and TNFα signaling to be downregulated in CML CD34\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;1G-H \u003cb\u003eand S2D\u003c/b\u003e). Together, these results suggest that in CML LSPCs, immune response, inflammation, and cytokine signaling were downregulated. Gene ontology analysis of significantly downregulated genes revealed the genes associated with positive regulation of cytokine production, Focal adhesion, and inhibitory MHCI receptor activity to be downregulated (Fig.\u0026nbsp;1I \u003cb\u003eand S2E-F)\u003c/b\u003e.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eLSPCs- Specific gene signature\u003c/h2\u003e \u003cp\u003eLeukemic stem/progenitor cell (LSPC)-specific gene signature in chronic myeloid leukemia (CML) was evaluated by comparing transcriptomic data generated in this study with two publicly available CML CD34\u003csup\u003e+\u003c/sup\u003e datasets (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). This integrative analysis was done to reduce dataset-specific biases and to identify robust, reproducible gene expression changes characteristic of CML LSPCs. Consistent upregulation of genes involved in heme metabolism, porphyrin metabolism, and heme biosynthesis pathways was observed across datasets (Fig.\u0026nbsp;2A-B\u003cb\u003e).\u003c/b\u003e These pathways are crucial for iron homeostasis and redox regulation, processes essential for cellular proliferation and survival in leukemic progenitors. In contrast, genes commonly downregulated were enriched for biological processes related to cellular and leukocyte homeostasis (Fig.\u0026nbsp;2C), indicating disruption of normal hematopoietic regulatory mechanisms. A core set of 28 genes was identified as consistently upregulated, while 18 genes were consistently downregulated across all datasets (Fig.\u0026nbsp;2D), implicating dysregulated iron metabolism as a key feature of CML LSPCs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptome analysis predicted unique cell surface marker expression in CML CD34\u003csup\u003e+\u003c/sup\u003e cells:\u003c/h2\u003e \u003cp\u003eWe then evaluated alterations in cell surface markers in CML CD34\u003csup\u003e+\u003c/sup\u003e cells, as these changes can serve as biomarkers and help distinguish between LSPCs and HSCs. Our analysis revealed that in CML CD34\u003csup\u003e+\u003c/sup\u003e cells, the expressions of \u003cem\u003eCD200R1, CD276, CD96, and CD36\u003c/em\u003e was increased, while \u003cem\u003eCD79A, CD37\u003c/em\u003e, and \u003cem\u003eCD22\u003c/em\u003e decreased compared to mPB donor CD34\u003csup\u003e+\u003c/sup\u003e cells. While CD36 has been previously reported as a CML LSC marker, the upregulation of CD96, a recognized marker for AML LSCs, is noteworthy and requires further validation to distinguish CML LSPCs from HD HSPCs. (\u003cb\u003eFigure S3A\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCML CD34\u003csup\u003e+\u003c/sup\u003e cells showed significantly increased expression of serine protease Cathepsin G:\u003c/h2\u003e \u003cp\u003eAmong the significantly upregulated genes in CML CD34\u003csup\u003e+\u003c/sup\u003e cells, one of the top targets was the serine protease family. Notably, serine proteases such as Cathepsin G (\u003cem\u003eCTSG)\u003c/em\u003e, Cathepsin L (\u003cem\u003eCTSL)\u003c/em\u003e, and neutrophil elastase (\u003cem\u003eELANE\u003c/em\u003e) were significantly upregulated in CML CD34\u003csup\u003e+\u003c/sup\u003e cells. In addition, ribonuclease (\u003cem\u003eRNASE)\u003c/em\u003e (\u003cb\u003eFigure S3B\u003c/b\u003e) was also upregulated in CML CD34\u003csup\u003e+\u003c/sup\u003e cells.\u003c/p\u003e \u003cp\u003eSince the validation of targets obtained from gene expression data can pose a substantial challenge, mainly due to the low abundance of CD34\u0026thinsp;+\u0026thinsp;cells in both CML and mPB donors, we utilized NanoString technology, which can generate high-quality gene expression results even from 100 ng of RNA. Based on Bloodspot (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://servers.binf.ku.dk/bloodspot/\u003c/span\u003e\u003cspan address=\"https://servers.binf.ku.dk/bloodspot/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e and previous studies (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), a 21-gene panel of potentially differentially expressed genes was custom-designed. The expression of these 21 genes was evaluated in an independent cohort of CML-CD34\u003csup\u003e+\u003c/sup\u003e (n\u0026thinsp;=\u0026thinsp;39) and mPB-CD34\u003csup\u003e+\u003c/sup\u003e cells (n\u0026thinsp;=\u0026thinsp;9). We observed significant upregulation of \u003cem\u003eCTSG\u003c/em\u003e and \u003cem\u003eELANE\u003c/em\u003e (Fig.\u0026nbsp;3A) and considerable downregulation of \u003cem\u003eCDH2, S100A8, S100A9\u003c/em\u003e, and \u003cem\u003eRXRA\u003c/em\u003e in CML CD34\u003csup\u003e+\u003c/sup\u003e cells compared to the mPB CD34\u003csup\u003e+\u003c/sup\u003e (Fig.\u0026nbsp;3B), in line with the transcriptomic results. The result was further corroborated by an immunoblotting experiment showing increased CTSG protein expression in CML CD34\u003csup\u003e+\u003c/sup\u003e cells compared to mPB CD34\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;3F). Further, we tested the expression of \u003cem\u003eCTSG\u003c/em\u003e in CML primary samples using q-RT-PCR. There was significant upregulation of \u003cem\u003eCTSG\u003c/em\u003e in treatment-na\u0026iuml;ve CML patients compared to healthy donor granulocytes, as depicted in \u003cb\u003eFig.\u0026nbsp;3C\u003c/b\u003e. However, no significant difference in \u003cem\u003eCTSG\u003c/em\u003e expression was observed between CML patients who achieved major molecular response (MMR) and those who did not (Fig.\u0026nbsp;3D), suggesting that \u003cem\u003eCTSG\u003c/em\u003e expression may not directly correlate with response to TKI therapy or disease prognosis in CML. Further, we tested \u003cem\u003eCTSG\u003c/em\u003e expression in CML patients presenting at blast crisis (BC) compared to those in the chronic phase (CP). \u003cem\u003eCTSG\u003c/em\u003e expression was markedly reduced in CML blast crisis patients (Fig.\u0026nbsp;3E), since CTSG is mainly present in the azurophilic granules in the myeloid compartment and hence has low expression in blasts.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMolecular inhibition of CTSG reduces colony-forming capacity and leukemogenic potential\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eTo evaluate the functional significance of \u003cem\u003eCTSG\u003c/em\u003e in CML, we carried out CTSG knockdown (\u003cem\u003eCTSG\u003c/em\u003e KD) in the CML cell line. The experimental design is outlined in \u003cb\u003eFig.\u0026nbsp;4A.\u003c/b\u003e Our initial screening of CML cell lines revealed that the EM-2 cell line expressed the highest level of CTSG protein expression (Fig.\u0026nbsp;4B). Hence, we transduced EM-2 cells with doxycycline (dox)-inducible \u003cem\u003eCTSG\u003c/em\u003e shRNAs (\u003cem\u003eCTSG\u003c/em\u003e KD). After five days of dox induction, \u003cem\u003eCTSG\u003c/em\u003e KD cells showed a marked reduction in the CTSG protein expression compared with the scramble, confirming knockdown (Fig.\u0026nbsp;4C). We assessed the effect of knockdown in the proliferation and colony-forming capacity. EM-2 CTSG KD cells did not show any difference in proliferation (\u003cb\u003eFigure S4A\u003c/b\u003e) but exhibited a reduced colony-forming capacity, supporting the possible role of \u003cem\u003eCTSG\u003c/em\u003e in the maintenance of CML cell stemness (Fig.\u0026nbsp;4D).\u003c/p\u003e \u003cp\u003eTo investigate the impact of CTSG suppression of self-renewal potential \u003cem\u003ein-vivo\u003c/em\u003e, we transplanted EM-2 CTSG KD cells, with and without doxycycline induction, into sublethally irradiated NSG mice (Fig.\u0026nbsp;4E).CTSG knockdown significantly reduced the repopulating potential leading to increased overall survival of the mice compared to the control group (Fig.\u0026nbsp;4F-G). These findings suggest that CTSG may be a promising target for therapeutic intervention in CML.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eTesting pharmacological inhibition of CTSG\u003c/h2\u003e \u003cp\u003eJin et al. demonstrated that Ponasterone A treatment reduces CTSG protein expression in an AML cell line. Based on this finding, we investigated the effects of Ponasterone A on CML CD34\u003csup\u003e+\u003c/sup\u003e cells and mPB CD34\u003csup\u003e+\u003c/sup\u003e(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Treatment with Ponasterone A consistently led to a dose-dependent decrease in CTSG protein expression (Fig.\u0026nbsp;5A) and a subsequent reduction in colony-forming capacity in CML CD34\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;5B). In contrast, Ponasterone A treatment did not have any effect on the colony-forming capacity of mPB CD34\u003csup\u003e+\u003c/sup\u003e (\u003cb\u003eFigure S4B\u003c/b\u003e). These results suggest that pharmacological inhibition of CTSG may reduce the colony-forming capacity of CML CD34\u0026thinsp;+\u0026thinsp;cells, indicating a potential role for CTSG in the self-renewal of CML cells. Further investigations are warranted to elucidate the precise mechanisms by which CTSG influences leukemogenesis and to determine the specific downstream pathways or cellular processes affected by its suppression. Understanding the molecular and cellular consequences of CTSG knockdown in the context of CML will provide valuable insights for developing targeted therapies to disrupt the leukemogenic potential of CML cells.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eChronic myeloid leukemia (CML) is driven by leukemic stem/progenitor cells (LSPCs) that sustain disease progression and resistance to therapy. Understanding the molecular signature of these cells is critical for developing targeted treatment.\u0026nbsp;Transcriptomic profiling of CML CD34\u003csup\u003e+\u003c/sup\u003e cells has revealed notable alterations in gene expression, with 1,563 genes upregulated and 1,400 genes downregulated. Pathway enrichment analysis has pinpointed significant changes in genes associated with the cell cycle, the Fanconi anemia pathway, and metabolism in CML CD34\u003csup\u003e+\u0026nbsp;\u003c/sup\u003ecells, indicating a shift towards enhanced oxidative phosphorylation (OxPHOS), akin to previous findings\u0026nbsp;(9). Upregulation of genes such as FANCI, \u003cem\u003eFANCM\u003c/em\u003e,\u003cem\u003e\u0026nbsp;FANCB\u003c/em\u003e, \u003cem\u003eFANCG\u003c/em\u003e, and \u003cem\u003eFANCD2\u003c/em\u003e within the Fanconi anemia pathway suggests potential enhancement of DNA repair mechanisms. Similarly, metabolic genes related to ETC-complex I and fatty acid \u0026beta; oxidation exhibited significant upregulation, indicating altered metabolism in CML CD34+ cells. These findings underscore the dysregulated metabolism and enhanced DNA repair mechanisms characteristic of CML CD34\u003csup\u003e+\u003c/sup\u003e cells. The dysregulation of the Fanconi anemia pathway has been previously implicated in CML CD34\u003csup\u003e+\u003c/sup\u003e cells, leading to chromosome instability\u0026nbsp;(17). Additionally, altered mitochondrial metabolism, as evidenced by the upregulation of ETC-I genes, can exacerbate oxidative stress and DNA damage, further contributing to genomic instability\u0026nbsp;(18).\u003c/p\u003e\n\u003cp\u003eFurthermore, changes in cell surface marker expression have been observed in CML CD34\u003csup\u003e+\u003c/sup\u003e cells, with upregulation of \u003cem\u003eCD200R1, CD276, CD96,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;CD36\u003c/em\u003e and downregulation of \u003cem\u003eCD79A\u003c/em\u003e, \u003cem\u003eCD37\u003c/em\u003e and \u003cem\u003eCD22\u003c/em\u003e. Notably, CD36 upregulation in CD34+CD38low CML cells has been linked to reduced responsiveness to imatinib, yet increased vulnerability to antibody-based therapies\u0026nbsp;(19). CD96, a recognized LSC marker in AML, and CD276, an immune checkpoint inhibitor, a marker highly expressed in monocytic AML, are therapeutically relevant targets. Preclinical studies have shown promising results in the development of CAR-T therapies targeting CD276\u0026nbsp;(20\u0026ndash;22).\u0026nbsp;The downregulation of \u003cem\u003eCD79A\u003c/em\u003e, \u003cem\u003eCD37\u003c/em\u003e, and \u003cem\u003eCD22\u003c/em\u003e suggests perturbed B-cell signaling and activation in CML, contributing to the selective accumulation of myeloid cells and a reduction in the lymphoid lineage. While previous studies have reported elevated expression of CD26 and CD25 in CML LSCs\u0026nbsp;(23,24), no significant differences were observed in our study, possibly due to the use of CML CD34\u003csup\u003e+\u003c/sup\u003e cells rather than the more primitive CML CD34\u003csup\u003e+\u003c/sup\u003eCD38\u003csup\u003e-\u003c/sup\u003e subset.\u003c/p\u003e\n\u003cp\u003eMoreover, our results demonstrate significant upregulation of serine proteases in CML CD34\u003csup\u003e+\u003c/sup\u003e cells, implicating their potential role in LSC survival. Serine proteases, including proteinase 3 (P3) and neutrophil elastase (ELANE), have been implicated in cancer development and progression, with potential immunomodulatory effects\u0026nbsp;(25). Further validation confirmed the upregulation of CTSG and ELANE, primary granule proteins, and the downregulation of S100A8/A9, secondary granule proteins. CTSG has been associated with immune response regulation and may serve as a potential therapeutic target in CML\u0026nbsp;(24).\u0026nbsp;In addition, Molecular inhibition of \u003cem\u003eCTSG\u003c/em\u003e impaired self-renewal properties in a CDX mouse model in vivo. Pharmacological inhibition of CTSG showed a dose-dependent reduction of CTSG and reduced colony-forming capacity in primary CD34\u003csup\u003e+\u003c/sup\u003e CML cells.\u0026nbsp;These results suggest the potential role of CTSG in the maintenance of CML LSPCs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe compared our datasets with publicly available datasets to identify the LSPC-specific gene signature. This comparative approach reduces dataset-specific biases and highlights robust, reproducible changes in gene expression. Genes involved in heme metabolism, porphyrin metabolism, and heme biosynthesis were consistently upregulated in CML CD34\u003csup\u003e+\u003c/sup\u003e cells. These pathways are essential for iron handling and redox biology, which are critical for cell proliferation and survival. Genes related to cellular and leukocyte homeostasis were commonly downregulated, suggesting impaired normal hematopoietic regulation in leukemic progenitors. The identification of 28 upregulated and 18 downregulated genes across datasets forms a robust molecular signature of CML LSPCs. This signature implicates dysregulated iron metabolism as a hallmark of these cells. Iron and heme metabolism are tightly linked to cellular energy production, oxidative stress, and DNA synthesis. Dysregulation of these pathways may provide CML LSPCs with survival advantages or contribute to therapy resistance. Targeting iron metabolism could selectively affect leukemic stem cells while sparing normal hematopoietic cells.\u003c/p\u003e\n\u003cp\u003eIn summary, Global transcriptomic analysis of CML vs. normal CD34\u003csup\u003e+\u003c/sup\u003e cells provided valuable insights into the molecular intricacies of CML, highlighting altered pathways, cell surface markers, and the potential role of \u003cem\u003eCTSG\u003c/em\u003e in LSPC survival. These findings offer a foundation for future research to develop targeted therapeutic strategies to disrupt these survival mechanisms and improve treatment outcomes for CML patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eESB, SI, BMR, AM, and MRA contributed to setting up all the in-vitro and in-vivo experiments, and transcriptome experiments, including analysis. AA and VM provided primary patient material and clinical response results of the patients. SRV provided input on data analysis and troubleshooting in-vitro experiments.PB procured research funding, designed the study, analyzed data, and provided a critical review of the manuscript. All the authors reviewed the manuscript, suggested comments, and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eTechnical assistance provided by Ms. Sangeetha is gratefully acknowledged. We thank the staff of the flow cytometry facility of the Centre for Stem Cell Research, Bagayam, Vellore, for their help.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eData Availability statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are available in the gene expression omnibus (GEO) repository, GSE315681.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConflict-of-interest disclosure\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe authors declare no conflict of interest to disclose.\u003c/em\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChu, S. et al. 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Cathepsin G Is Expressed by Acute Lymphoblastic Leukemia and Is a Potential Immunotherapeutic Target. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 1975 (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8580579/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8580579/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChronic myeloid leukemia (CML) stem and progenitor cells (LSPCs) are not eliminated by tyrosine kinase inhibitors, resulting in disease relapse or progression. Transcriptomic profiling identified Cathepsin G (\u003cem\u003eCTSG\u003c/em\u003e), a serine protease, as one of the top significantly upregulated targets in primary CML CD34\u0026thinsp;+\u0026thinsp;cells compared to CD34\u0026thinsp;+\u0026thinsp;cells from healthy donors (Log2 FC, p-value). Further, molecular inhibition of \u003cem\u003eCTSG\u003c/em\u003e reduced the self-renewal capacity and diminished disease burden in vivo in a CDX mouse model, highlighting \u003cem\u003eCTSG\u003c/em\u003e as a promising therapeutic target in CML. Pharmacological inhibition of CTSG demonstrated a dose-dependent decrease in \u003cem\u003eCTSG\u003c/em\u003e expression and colony-forming capacity only in CML CD34\u0026thinsp;+\u0026thinsp;cells. This study offers comprehensive insights into the gene expression landscape of CML LSPCs, highlighting the functional significance of CTSG in disease progression. Targeting \u003cem\u003eCTSG\u003c/em\u003e could be a novel therapeutic strategy for CML treatment by disrupting the leukemogenic potential and improving therapeutic efficacy.\u003c/p\u003e","manuscriptTitle":"Targeting Cathepsin-G to Overcome Leukemic Stem Cell Persistence in Chronic Myeloid","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-04 12:43:43","doi":"10.21203/rs.3.rs-8580579/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-24T19:05:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-13T09:26:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-10T08:09:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"212648139792329333500622112699693960310","date":"2026-02-24T01:34:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50810096679546959727339827726164473886","date":"2026-02-22T13:20:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"318353519383197153080223886697343812837","date":"2026-02-18T16:04:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"91462096049993434105154451223514320323","date":"2026-02-16T13:34:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"38048462112107763243132847699382108015","date":"2026-02-12T06:41:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-02T13:31:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-01T19:29:23+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-30T10:10:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-30T05:47:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-01-30T05:38:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2a055dfb-d7fa-4fa6-9fbc-27baa4267933","owner":[],"postedDate":"February 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":62205324,"name":"Biological sciences/Cancer"},{"id":62205325,"name":"Biological sciences/Cell biology"},{"id":62205326,"name":"Biological sciences/Drug discovery"}],"tags":[],"updatedAt":"2026-04-30T20:08:12+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-04 12:43:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8580579","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8580579","identity":"rs-8580579","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}