scholarly journals Characterization of Leukemic Stem Cells Heterogeneity in Chronic Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4140-4140
Author(s):  
Rebecca Warfvinge ◽  
Mikael Sommarin ◽  
Parashar Dhapola ◽  
Ulrich Pfisterer ◽  
Linda Geironson Ulfsson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Single Cell ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Single Cell Analysis ◽  
Therapy Response ◽  
Surface Markers ◽  
Sequencing Analysis ◽  
Leukemic Stem Cells

In chronic myeloid leukemia (CML), a rare subset of leukemic stem cells (LSC) persists in patients responding to conventional tyrosine kinase inhibitor (TKI) therapy. The failure to eradicate these LSCs results in indefinite therapy dependence and a risk of leukemic relapse. However, the conventional LSC compartment (Lin-CD34+CD38-) is highly heterogeneous where only a subpopulation is believed to be functional, TKI-insensitive LSCs. Previously, using single-cell gene expression analysis we characterized the heterogeneity within the LSC population (Lin-CD34+CD38-) in CML patients using a selected panel of 96 primers. Interestingly, by comparing LSC heterogeneity at diagnosis with the heterogeneity following 3 months of TKI therapy we uncovered a therapy-insensitive, quiescent subpopulation, which could be isolated at high-purity using a combination of the surface markers: Lin-CD34+CD38-CD45RA-cKIT-CD26+ (Warfvinge, Geironson, Sommarin et al., 2017). Here, we expand the single-cell analysis of CML LSC populations to include combined immunophenotype-/RNA sequencing analysis (CITE-seq). CITE-seq allows for unbiased, further in-depth transcriptome analysis as wells as immunophenotypic characterization by pre-staining cells with a panel of DNA-barcoded antibodies prior to sequencing. DNA-barcoded antibodies convert the protein expression into readable sequences through unique oligo-conjugates as identifiers. Using CITE-seq with a panel of 44 distinct surface markers designed to immunophenotypically differentiate between stem/progenitors cells and leukemic clones we simultaneously characterize the molecular and immunophenotypic heterogeneity within Lin-CD34+/Lin-CD34+CD38- CML stem/progenitor compartment at diagnosis. Additionally by comparing the LSCs transcriptome from patients with different therapeutic outcome after 12 months of therapy we describe how differences in heterogeneity and the presence of immunophenotypic therapy-insensitive LSCs at diagnosis (Lin-CD34+CD38-CD45RA-cKIT-CD26+) contribute to therapy response. Disclosures Richter: Novartis: Consultancy; Pfizer: Consultancy, Research Funding.

scholarly journals Therapeutic inhibition of FcγRIIb signaling targets leukemic stem cells in chronic myeloid leukemia

Leukemia ◽  
2020 ◽  
Vol 34 (10) ◽  
pp. 2635-2647
Author(s):  
Oliver Parting ◽  
Samantha Langer ◽  
Maja Kim Kuepper ◽  
Caroline Wessling ◽  
Shaoguang Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Therapeutic Approach ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Leukemic Stem Cells ◽  
Downstream Signaling ◽  
Targeted Inhibition ◽  
Treatment Free Remission

Abstract Despite the successes achieved with molecular targeted inhibition of the oncogenic driver Bcr-Abl in chronic myeloid leukemia (CML), the majority of patients still require lifelong tyrosine kinase inhibitor (TKI) therapy. This is primarily caused by resisting leukemic stem cells (LSCs), which prevent achievement of treatment-free remission in all patients. Here we describe the ITIM (immunoreceptor tyrosine-based inhibition motif)-containing Fc gamma receptor IIb (FcγRIIb, CD32b) for being critical in LSC resistance and show that targeting FcγRIIb downstream signaling, by using a Food and Drug Administration-approved BTK inhibitor, provides a successful therapeutic approach. First, we identified FcγRIIb upregulation in primary CML stem cells. FcγRIIb depletion caused reduced serial re-plaiting efficiency and cell proliferation in malignant cells. FcγRIIb targeting in both a transgenic and retroviral CML mouse model provided in vivo evidence for successful LSC reduction. Subsequently, we identified BTK as a main downstream mediator and targeting the Bcr-Abl-FcγRIIb-BTK axis in primary CML CD34+ cells using ibrutinib, in combination with standard TKI therapy, significantly increased apoptosis in quiescent CML stem cells thereby contributing to the eradication of LSCs.. As a potential curative therapeutic approach, we therefore suggest combining Bcr-Abl TKI therapy along with BTK inhibition.

scholarly journals High Throughput Immunophenotyping and Expression Profiling at Single Cell Level Reveal BCR-ABL1 Dependent Surface Markers of Chronic Myeloid Leukemia Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2920-2920
Author(s):  
Marianna Romzova ◽  
Dagmar Smitalova ◽  
Peter Taus ◽  
Jiri Mayer ◽  
Martin Culen
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Expression Profiling ◽  
Myeloid Leukemia ◽  
Single Cells ◽  
Transcript Level ◽  
Surface Expression ◽  
Surface Markers ◽  
Cell Level ◽  
Tki Treatment

BACKGROUND: Bcr-abl1 oncogene targeted treatment with tyrosine kinase inhibitors (TKI) showed an impressive efficacy against proliferating chronic myeloid leukemia (CML) cells. However, rapid relapses in more than half of CML patients after discontinuation of the treatment suggest a presence of quiescent leukemic stem cells inherently resistant to BCR-ABL1 inhibition. Understanding the heterogeneity of CML stem cell compartment is crucial for preventing the treatment failure. Specificity of already established leukemic stem cell (LSC) markers has been tested mainly in bulk CD34+CD38- populations at diagnosis. Phenotypes and molecular signatures of therapy resistant BCR ABL1 positive stem cells is however yet to be established. AIMS: Identification of BCR-ABL1 dependent LSC markers at single cell level by direct comparison their surface and transcript expression with the levels and the presence of BCR-ABL1 transcript at diagnosis and after administration of TKI treatment. METHODS: Total number of 375 cells were obtained from bone marrow and peripheral blood of 4 chronic phase CML patients. Cells were collected prior any treatment and three months after TKI treatment initiation. Normal bone marrow cells and BCR-ABL1 positive K562 cell line were used as controls. Indexed immuno-phenotyping and sorting of CD34+CD38- single cells was performed using a panel of 11 specific surface markers. Collected single cells were lysed and cDNA was enriched for 11 targets using 22 cycle pre-amplification. Expression profiling was carried on SmartChip real-time PCR system (Takara Bio) detecting following genes: BCR-ABL1, CD26, CD25, IL1-Rap, CD56, CD90, CD93, CD69, KI67, and control genes GUS and HPRT. Unsupervised clustering was performed using principal component analysis (PCA). Correlations were measured by Spearman rank method. RESULTS: At diagnosis, majority of BCR-ABL1+ C34+CD38- stem cells co-express IL1-Rap, CD26, and CD69 on their surface (88%, 82%, 78% overlap). Only 56% of BCR-ABL1+ cells positive for aforementioned markers co-express CD25, 28% CD93 and 16% CD56. The expression of these markers could also be detected in 4-11% of BCR-ABL1- cell, although this could be technical inaccuracy caused by the single cell profiling. CD90 marker did not show any correlation with BCR-ABL1 expression. At transcript level the expression of IL-1Rap, CD26, CD25 and CD56 was observed in 62%, 52% 45% and 16% BCR-ABL1+ cells, and up to 7% of BCR-ABL1- cells. CD69 expression was observed in 90% of BCR-ABL+ cells at transcript level, but also in 71% BCR-ABL- cells. BCR-ABL1 independent expression was observed for cKIT. (60% vs. 76 % in positive vs negative). Finally proliferation marker KI67 was expressed only in 6% of the BCR-ABL1+ cells. PCA analysis divided cells into several distinct clusters with BCR-ABL1 as the main contributor, and cKIT, CD69 and CD26, IL-1RAP as other significant factors. Interestingly BCR-ABL1+ cells collected during TKI treatment showed persistent surface expression of IL-1Rap and CD26, while CD56, CD69 and CD93 were only on part of the BCR-ABL1+ cells. CD25 was significantly deregulated during TKI treatment. CONCLUSION: At diagnosis up to 80% of LSC co-express 3 specific surface markers - IL-1RAP, CD26 and CD69. Variable portion of LSC co-express additional markers such are CD25, CD56 and CD93. During TKI treatment the surface expression of majority of markers is decreased, where the best correlated LSC marker is IL-1Rap, followed by CD26 and CD69. CD56 marker seems to persist in the same proportion of cells while CD25 disappears. cKIT is highly expressed in normal BM and HSC from CML patients, but also in some LSC. CD34+CD38- cells show non-proliferating phenotype. Disclosures Mayer: AOP Orphan Pharmaceuticals AG: Research Funding.

Dipeptidylpeptidase IV (CD26) defines leukemic stem cells (LSC) in chronic myeloid leukemia

Blood ◽  
2014 ◽  
Vol 123 (25) ◽  
pp. 3951-3962 ◽  
Author(s):  
Harald Herrmann ◽  
Irina Sadovnik ◽  
Sabine Cerny-Reiterer ◽  
Thomas Rülicke ◽  
Gabriele Stefanzl ◽  
...  
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Specific Marker ◽  
Leukemic Stem Cells ◽  
Dipeptidylpeptidase Iv ◽  
Key Points

Key Points DPPIV (CD26) is a new specific marker of CML LSC that aids CML diagnostics and the measurement, characterization, and purification of LSC. DPPIV on CML LSC degrades SDF-1 and thereby promotes the niche-escape of LSC, which may contribute to extramedullary myeloproliferation in CML.

Kinase-inhibitor–insensitive cancer stem cells in chronic myeloid leukemia

2014 ◽  
Vol 14 (3) ◽  
pp. 287-299 ◽  
Author(s):  
Alessandro Morotti ◽  
Cristina Panuzzo ◽  
Carmen Fava ◽  
Giuseppe Saglio
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Cancer Stem Cells ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor

Induced Pluripotent Stem Cells (iPSC) From Chronic Myeloid Leukemia: Study of BCR-ABL Addiction and Effect of Tyrosine Kinase Inhibitors,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3754-3754 ◽  
Author(s):  
Aurélie Bedel ◽  
Francois Moreau-Gaudry ◽  
Jean- Max Pasquet ◽  
Miguel Taillepierre ◽  
Éric Lippert ◽  
...  
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Western Blot ◽  
Blood Cells ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Leukemic Stem Cells ◽  
Rt Pcr ◽  
Reprogramming Factors

Abstract Abstract 3754 The tyrosine kinase inhibitors (TKI) such as imatinib, by suppressing BCR-ABL oncogene activity, are an effective therapy for chronic myeloid leukemia disease (CML). However, the majority of patients achieving remission with TKI continue have molecular evidence of persistent disease. In addition, we have reported that for patients who achieved a sustained complete molecular remission, 60% of them relapse after discontinuation of imatinib. Various mechanisms have been proposed to explain disease persistence and disease recurrence. One of the hypotheses is that primitive leukemic stem cells can survive in the presence of TKI. Little is known about the stem cells survival due to technical difficulties (small and poorly defined primary populations). Understanding the mechanisms by which these cells survive to TKI therapy will be critical to devising strategy aimed to their elimination. We propose to generate iPSC derived from CD34+ blood cells isolated from CML patient (CML-iPSC), as a model for study leukemic stem cells survival in the presence of TKI and study the mechanism of TKI resistance of the stem cells. Primary CD34+ CML patient cells were transduced by 2 excisable lentiviral vectors (both flanked by two LoxP sites), one expressing three reprogramming factors (OCT4-SOX2-KLF4) and another one with c-MYC and a shRNA against TP53. Twenty-one days after co-transduction, CML-iPSC colonies were picked and five iPS clones were characterized (expression of pluripotency markers by RT-PCR (DPPA4, NANOG, CRIPTO) and immunofluorescence (NANOG, SSEA-4, TRA1-60)). Efficiency of reprogrammation was low compared to cord blood CD34+ control cells (0.01% vs 0.1%, respectively), and delayed (21 days vs 14 days). Philadelphia chromosome (Ph) positive was observed in 4/5 clones after cytogenetic analysis. Expression of BCR-ABL (Western-blot and RT-PCR) was present at various levels. Interestingly, 1/5 clone was generated from non-leukemic cell (Ph negative) and was used as internal control for the following function assays. We used these 5 CML-iPSC clones to study their behavior in presence of TKI. All CML-iPSC clones survived to escalating concentration of imatinib (0 to 20μM) and ponatinib (0 to 50nM) for 6 days. To understand if the CML-iPSC survival was due to resistance or independence mechanisms, we performed western blot analysis of TKI targets. BCR-ABL activity was inhibited under TKI exposure (dephosphorylations of BCR/ABL and of Crkl). In order to check whether survival was due to the expression of reprogramming factors, we excised the gene cassettes by an Adenovirus expressing CRE recombinase. After proviral excision and subcloning, excised CML-iPSC continued to survive to TKI exposure. Taken together, these results demonstrate that CML-iPSC survival do not depend on BCR-ABL (oncogene independence). Upon induction of hematopoietic differentiation, CML-iPSC were able to efficiently generate progenitors of hematopoietic lineages (up to 40% of CD45+) and colony forming units in methylcellulose. TKI effect on iPSC-derived hematopoietic progenitors, to analyze the putative recovery of TKI sensibility compared to primitive CML blood cells from the same patient, are in progress. We conclude that reprogrammation of CD34BCR-ABL+ cells from CML patient is possible and that CML-iPSC lost the BCR-ABL dependency and became resistant to TKI. A specific differentiated epigenetic cell state is probably needed to maintain BCR-ABL dependency. CML-iPSC can be used to study mechanisms by which leukemic stem cells survive to TKI therapy and is a promising tool for testing and screening new therapeutic target reducing leukemic stem cell survival. Disclosures: Mahon: Novartis Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria; Pfizzer: Honoraria.

scholarly journals Influence of Estrogen Treatment on ESR1+ and ESR1− Cells in ER+ Breast Cancer: Insights from Single-Cell Analysis of Patient-Derived Xenograft Models

Cancers ◽  
2021 ◽  
Vol 13 (24) ◽  
pp. 6375
Author(s):  
Hitomi Mori ◽  
Kohei Saeki ◽  
Gregory Chang ◽  
Jinhui Wang ◽  
Xiwei Wu ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Therapy Response ◽  
Sequencing Analysis ◽  
Gene Set ◽  
Patient Derived Xenograft ◽  
Potential Marker ◽  
The Impact

A 100% ER positivity is not required for an endocrine therapy response. Furthermore, while estrogen typically promotes the progression of hormone-dependent breast cancer via the activation of estrogen receptor (ER)-α, estrogen-induced tumor suppression in ER+ breast cancer has been clinically observed. With the success in establishing estrogen-stimulated (SC31) and estrogen-suppressed (GS3) patient-derived xenograft (PDX) models, single-cell RNA sequencing analysis was performed to determine the impact of estrogen on ESR1+ and ESR1– tumor cells. We found that 17β-estradiol (E2)-induced suppression of GS3 transpired through wild-type and unamplified ERα. E2 upregulated the expression of estrogen-dependent genes in both SC31 and GS3; however, E2 induced cell cycle advance in SC31, while it resulted in cell cycle arrest in GS3. Importantly, these gene expression changes occurred in both ESR1+ and ESR1– cells within the same breast tumors, demonstrating for the first time a differential effect of estrogen on ESR1– cells. E2 also upregulated a tumor-suppressor gene, IL-24, in GS3. The apoptosis gene set was upregulated and the G2M checkpoint gene set was downregulated in most IL-24+ cells after E2 treatment. In summary, estrogen affected pathologically defined ER+ tumors differently, influencing both ESR1+ and ESR1– cells. Our results also suggest IL-24 to be a potential marker of estrogen-suppressed tumors.

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