A Pan-BCL2 Inhibitor Renders Bone-Marrow-Resident Human Leukemia Stem Cells Sensitive to Tyrosine Kinase Inhibition

Abstract Activating mutations of the FLT3 receptor by internal tandem duplication (FLT3-ITD) are present in 30% of all cases of acute myeloid leukemia (AML) and are associated with poor prognosis. FLT3-ITD mutations are present in leukemic stem/progenitor cells and induce ligand-independent downstream signaling promoting oncogenesis through pathways involved in proliferation, differentiation and survival, making the mutated receptor an attractive therapeutic target for tyrosine kinase inhibition. Although tyrosine kinase inhibitors have been shown to be cytotoxic to FLT3-ITD+ leukemic blasts, the effects on more primitive leukemic stem cells have not been studied in detail. We examined the effect of the tyrosine kinase inhibitor SU5614 on leukemic CD34+ stem/progenitor cells from patients with newly diagnosed normal karyotype AML with wild-type FLT3 or mutated FLT3-ITD receptor. SU5614 was chosen because initial experiments comparing SU5614, PKC412 and imatinib had shown that SU5614 was the most potent in inducing cell cycle arrest without significant apoptosis in normal CD34+ stem/progenitor cells. CD34+ cells were isolated from bone marrow of AML patients at diagnosis by density gradient centrifugation and magnetic bead isolation. Cells were cultured for four days in serum-free medium with growth factors in the presence or absence of SU5614 (5 uM) in suspension culture or in stroma-contact cultures. Hematopoietic activity was assessed in colony-forming assays. Overall, untreated CD34+FLT3-ITD+ leukemic progenitors cells formed significantly fewer CFU than CD34+FLT3-WT leukemic progenitors. However, the percentage of more primitive LTC-IC was higher in FLT3-ITD+ samples. SU5614 induced cell cycle arrest in all FLT3-ITD+ as well as FLT3-WT samples whereas apoptosis was variable. FLT3-ITD+ committed progenitor cells were effectively reduced by SU5614 treatment in suspension culture while stroma contact exerted a significant protective effect. In contrast, committed progenitors from FLT3-WT AML were less susceptible to tyrosine kinase inhibition but also protected by adhesion to stroma. More importantly, primitive LTC-IC from FLT3-ITD+ AML were selectively spared from tyrosine kinase inhibition. Additional stromal contact led to expansion of LTC-IC in the presence of SU5614. PCR from single hematopoietic colonies of stromal contact cultures revealed both WT and FLT3-ITD products after treatment with SU5614, indicating LTC-IC were of leukemic origin. To further elucidate the mechanism by which stromal contact selectively protects FLT3-ITD+ LTC-IC, leukemic cell lines harboring either FLT3-ITD (MV4-11) or FLT3-WT (RS 4;11) were studied. As expected, SU5614 effectively inhibited constitutively active FLT3 in MV4-11 as well as ligand activated FLT3 in RS 4;11 cell lines independent of stromal contact. However, inhibition of downstream Akt activation by SU5614 in MV4-11 cells was completely abrogated in the presence of stroma. In contrast, stromal contact had no effect on Akt activation in FLT3-WT RS 4;11 cells. Activation of downstream Erk and Stat5 and inhibition by SU5614 were not affected by stromal contact in either cell line. In conclusion, our data suggest activation of alternate signaling pathways in FLT3+ leukemic stem cells allowing escape from dependence on FLT3 signaling and subsequently tyrosine kinase inhibition. In addition, protection of leukemic FLT3-ITD+ LTC-IC is mediated by stromal contact.

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Oncogene Cooperativity Analysis Reveals a Novel Set of Genes That Regulate the In Vivo Growth and Survival of Leukemia Stem Cells

Blood ◽

10.1182/blood.v118.21.553.553 ◽

2011 ◽

Vol 118 (21) ◽

pp. 553-553

Author(s):

John M Ashton ◽

Marlene Balys ◽

Sarah Neering ◽

Glenn Cowley ◽

David E. Root ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Leukemic Cell ◽

Gene Signature ◽

Leukemic Cells ◽

Leukemia Stem Cells ◽

Human Leukemia ◽

Normal Cells ◽

In Vivo Growth

Abstract Abstract 553 In order to increase our understanding of key biological properties governing the development of leukemia stem cells (LSCs), we employed a novel gene identification strategy based on cooperation between initiating oncogenes. Previous studies have demonstrated that genes whose expression is regulated in a synergistic manner as a consequence of two cooperating oncogenes (termed “cooperativity response genes”, or CRGs) are highly enriched for activity in tumor formation. Further, in contrast to the thousands of genes identified by differential expression analyses of normal vs. leukemic cell populations, CRGs represent a much smaller subset of targets; thereby, providing a defined set of genes to investigate. We adapted the CRG strategy to identify synergistically regulated genes in primitive leukemic cells. Using a mouse model of myeloid blast crisis leukemia induced through the cooperation of BCR-ABL and NUP98-HOXA9, we performed genome-wide transcriptional profiling comparing hematopoietic cells expressing each translocation alone or in combination. Using this system, we were able to model the genetic alterations induced as normal cells progressed towards LSC transformation, identifying 72 CRGs (50 aberrantly up-regulated and 22 down-regulated) with potential importance in leukemia development. To investigate the relevance of these CRGs in leukemia biology, an RNAi screen approach was employed. Primary leukemic progenitors were purified and transduced with a custom lentiviral RNAi library and subsequently transplanted into recipient animals to assess the engraftment potential upon perturbation of the individual CRGs. Our findings demonstrate that knock-down of expression in 35 of 50 (70%) leukemia CRGs reduced in vivo growth of primitive leukemia, a finding that was independently validated through single gene perturbation of several genes that scored in the RNAi screen (GJB3, EphA3, PMP22, Serinc2, SerpinB2, and CP). In particular, serpinB2, a gene that scored strongly in the RNAi analysis, was shown to directly effect the frequency of LSC in vivo. Given that the cooperative gene signature represented genes with many distinct cellular functions, we hypothesized that the CRG expression profile represents a key regulatory network in leukemia survival. To investigate our hypothesis we utilized the Broad Institute's Connectivity Map (CMAP) to identify pharmacological compounds with the ability to modulate multiple CRGs simultaneously. This analysis revealed that both Tyrophostin AG-825 (AG825) and 4-hydroxy-2-nonenol (4HNE) were predicted to reverse the gene expression induced as a consequence of leukemic transformation. To test the effect of these agents as selective toxicants to leukemia, we treated both normal and leukemia murine bone marrow cells with each compound. Both bulk and phenotypically primitive leukemic cells were eradicated in dose-responsive fashion upon treatment with either AG825 or 4HNE, while normal cells showed significantly reduced sensitivity. Progenitor function as measured by colony forming assays also showed a selective reduction in leukemia colony formation, suggesting that both these compounds are toxic to the majority of leukemic cell types. Interestingly, similar results were obtained when human normal and leukemic bone marrow specimens were treated with both drugs, suggesting the CRG signature represents an important class of genes with conserved function across species. To determine the level of conservation of the leukemia CRG signature between murine and human leukemia, we profiled eight normal and leukemic patient specimens for expression of the CRG signature. Of the 39 evaluable human CRG orthologs, 13 showed similar expression trends in human leukemia samples relative to normal controls. Intriguingly, both AG825 and 4HNE were predicted to inhibit this 13-gene signature by the CMAP database, suggesting that the compounds may act through these genes to influence leukemia cell death. Taken together, our findings demonstrate the importance of cooperative gene regulation in leukemogenesis and provide a novel platform for future research toward more effective therapeutic strategies to treat leukemia. Disclosures: No relevant conflicts of interest to declare.

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Epigenetic Reprogramming Sensitizes CML Stem Cells to Combined EZH2 and Tyrosine Kinase Inhibition

Cancer Discovery ◽

10.1158/2159-8290.cd-16-0263 ◽

2016 ◽

Vol 6 (11) ◽

pp. 1248-1257 ◽

Cited By ~ 65

Author(s):

Mary T. Scott ◽

Koorosh Korfi ◽

Peter Saffrey ◽

Lisa E.M. Hopcroft ◽

Ross Kinstrie ◽

...

Keyword(s):

Stem Cells ◽

Tyrosine Kinase ◽

Epigenetic Reprogramming ◽

Kinase Inhibition ◽

Tyrosine Kinase Inhibition

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Abstract 1929: Tyrosine kinase inhibition with sorafenib but not pazopanib enriches for sarcoma cancer stem cells in diverse models of soft tissue sarcoma

10.1158/1538-7445.am2014-1929 ◽

2014 ◽

Author(s):

Robert J. Canter ◽

Erik Ames ◽

Stephanie Mac ◽

Steve K. Grossenbacher ◽

Mingyi Chen ◽

...

Keyword(s):

Stem Cells ◽

Soft Tissue ◽

Cancer Stem Cells ◽

Soft Tissue Sarcoma ◽

Tyrosine Kinase ◽

Kinase Inhibition ◽

Tyrosine Kinase Inhibition

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Infliximab therapy together with tyrosine kinase inhibition targets leukemic stem cells in chronic myeloid leukemia

BMC Cancer ◽

10.1186/s12885-019-5871-2 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 1

Author(s):

Oliver Herrmann ◽

Maja Kim Kuepper ◽

Marlena Bütow ◽

Ivan G. Costa ◽

Iris Appelmann ◽

...

Keyword(s):

Stem Cells ◽

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Myeloid Leukemia ◽

Infliximab Therapy ◽

Leukemic Stem Cells ◽

Kinase Inhibition ◽

Tyrosine Kinase Inhibition

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Role of CXCL12-Expressing Mesenchymal Stromal Cell Niches in Maintaining Treatment-Resistant Leukemia Stem Cells

Blood ◽

10.1182/blood-2018-99-117762 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1291-1291

Author(s):

Puneet Agarwal ◽

Stephan Isringhausen ◽

Hui Li ◽

Andrew J Paterson ◽

Jianbo He ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Bone Marrow ◽

Tyrosine Kinase ◽

Expression Analysis ◽

Gene Expression Analysis ◽

Leukemia Stem Cells ◽

Hematopoietic Stem ◽

Tki Treatment

Abstract Chronic myeloid leukemia (CML) results from hematopoietic stem cell (HSC) transformation by the BCR-ABL tyrosine kinase. Tyrosine kinase inhibitors (TKI), although effective in inducing remissions in CML, fail to eradicate leukemia stem cells (LSC) which persist as source of relapse. LSC resistance to TKI-treatment occurs through kinase-independent mechanisms, which include alterations in intrinsic cell-regulatory mechanisms as well as signals from the bone marrow (BM) microenvironment that support LSC persistence. HSC have been shown to be regulated by C-X-C motif chemokine ligand 12 (CXCL12)-expressing bone marrow niches, but the nature and regulatory role of BM niches for LSC remains poorly understood.Here, we used CXCL12-GFP mice and CXCL12f/fmice crossed with Cre lines targeting specific CXCL12-expressing cells to investigate the contribution of CXCL12-expressing populations to LSC regulation. We found that targeted deletion of CXCL12 from Prx1+ mesenchymal stromal cells (MSC) reduced normal HSC numbers. In contrast, deletion of CXCL12 from Prx1+ MSC in the setting of CML, resulted in increased leukocytosis, neutrophilia, BM cellularity and LSC numbers, and reduced survival, compared to control CML mice. CXCL12 deletion from Prx1+ MSC was found to enhance LSC cycling. Despite increased cycling, the expanded LSC from these mice maintained their in vivo repopulating capacity. To evaluate the effect of CXCL12 deletion on LSC and MSC distribution, we performed 3D imaging of BM volumes from Prx1-Cre mice crossed with tdTomato reporter mice. CML development resulted in formation of large pathological tissue niches harboring an abnormally high density of MSCs as well as c-Kit+ leukemia progenitor cells. However, these MSC and leukemic progenitor clusters were not observed in Prx1-Cre+CXCL12fl/fl mice, indicating that formation of tissue structures with colocalized leukemic progenitors is dependent on CXCL12 expression in MSC. We performed gene expression analysis of LSC from Prx1-Cre+CXCL12f/f and Cre-negative mice. Gene expression analysis revealed enrichment of cell cycling and MYC related genes in CML LSC from Prx1-Cre+CXCL12f/f mice, and downregulation of Polycomb Repressive Complex 2 (PRC2) target genes, indicating increased PRC2 activity. We confirmed that EZH2 expression and H3K27 trimethylation were increased in LSC from Prx1-Cre+CXCL12f/f mice. Treatment with the EZH2 inhibitor, GSK 343, resulted in significant reduction in WBC, neutrophils, BM cellularity and LSC in Prx1-Cre+CXCL12f/f mice, but not control CML mice. These results support a role for increased PRC2 activity in LSC expansion in mice with CXCL12 deletion from Prx1+ MSC. We evaluated the effect of CXCL12 deletion from Prx1+ MSC on LSC sensitivity to treatment with the TKI Nilotinib. Treatment of Cre-negative CML mice with Nilotinib reduced WBC counts, spleen cellularity, and splenic LSC numbers, but did not reduce BM cellularity or LSC numbers. On the other hand, Nilotinib treatment significantly reduced BM cellularity and LSC numbers, and enhanced survival, in Prx1-Cre+CXCL12f/f CMLmice. Transplantation of BM from TKI-treated Prx1-Cre+CXCL12f/f mice to irradiated normal recipients resulted in significantly reduced long-term donor engraftment and donor LSC numbers compared to vehicle-treated mice. These results indicate that CXCL12 deletion from Prx1+ MSC leads to enhanced sensitivity of CML LSC to elimination by TKI treatment. In conclusion, our studies show that deletion of CXCL12 from Prx1+ MSC niches leads to loss of MSC clustering and colocalization with leukemic progenitors, and to loss of quiescence and expansion of LSC, dependent on enhanced PRC2 activity. CXCL12 deletion from MSC also increases TKI-mediated targeting of resistant, quiescent, self-renewing CML LSC. Strategies to inhibit CXCL12-mediated niche interactions represent a promising approach for LSC depletion to enhance opportunities for cures in CML. Disclosures No relevant conflicts of interest to declare.

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Leukemia Stem Cells in Murine MLL−AF9 AML Are Downstream Myeloid Lineage Cells That Have Acquired Stem−Like Biological and Genetic Properties.

Blood ◽

10.1182/blood.v108.11.771.771 ◽

2006 ◽

Vol 108 (11) ◽

pp. 771-771

Author(s):

Tim C.P. Somervaille ◽

Michael L. Cleary

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Leukemia Cell ◽

Leukemia Stem Cells ◽

Human Leukemia ◽

Self Renewal ◽

Myeloid Lineage ◽

Hematopoietic Stem ◽

Progenitor Compartment

Abstract An essential prerequisite for the development of more effective targeted therapies in AML is a characterization of the frequency and biological properties of leukemia stem cells (LSCs), which sustain the disease and mediate relapse. Previous studies have shown that hematopoietic stem cells, as well as progenitors, may be targeted by MLL oncoproteins to give rise to AML, however it was unclear whether these lineage negative cells also functioned as LSCs to sustain disease. To address this issue, we have identified and characterized LSCs in a somatic, genetic mouse model of leukemia that faithfully recapitulates many of the pathologic and clinical attributes of human leukemia initiated by the MLL−AF9 oncogene. In this model, CFCs in the bone marrow and spleen of leukemic mice were demonstrated to be LSCs, based on the observation that secondary transplantation of the progeny of individually isolated AML CFCs cultured in vitro for seven days or longer invariably resulted in transfer of short latency disease. These self−renewing cells were remarkably frequent, accounting for 25–30% of myeloid lineage cells at late−stage disease. Unexpectedly, they expressed mature myeloid lineage antigens (91.3 ± 3.8% Mac1+ Gr1+ immunophenotype, versus fewer than 0.2% lineage negative), placing them downstream of the known hematopoietic progenitor compartment. Furthermore, LSCs in this model generated a phenotypic, morphologic and functional leukemia cell hierarchy loosely defined by expression of c−kit. When compared with immortalized progenitors, LSCs exhibited a markedly enhanced ability to engraft secondary recipients, which was not due to differences in bone marrow homing, which was equivalently poor in the compared populations. Rather, LSCs exhibited an acquired ability to proliferate in response to stromal cell derived cytokines, an enhanced SDF1 induced chemotaxis, and increased proliferation in contact with OP9 stromal cells demonstrating that LSCs exhibit altered microenvironmental interactions by comparison with the oncogene immortalized CFCs that initiated the disease. Thus, the LSCs responsible for sustaining, expanding and regenerating MLL−AF9 AML are downstream myeloid lineage cells, outside of the normal stem and progenitor compartment. They have acquired an aberrant Hox−associated self−renewal program as well as other biologic features of hematopoietic stem cells. Our findings support a revision of the prevailing hypothesis that AML LSCs are always rare and solely located within the most immature bone marrow progenitor compartment. Furthermore, LSCs exhibit markedly different microenvironmental interactions, by comparison with cells simply immortalized by MLL−AF9, indicating that acquisition of sensitivity to stromal derived survival and proliferative signals is a critical feature of LSCs, in addition to their extensive self−renewal capabilities.

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