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Self-renewal is a feature of cancer and can be assessed by cell transplantation into immune-compromised or immune-matched animals. However, studies in zebrafish have been severely limited by lack of these reagents. Here, Myc-induced T-cell acute lymphoblastic leukemias (T-ALLs) have been made in syngeneic, clonal zebrafish and can be transplanted into sibling animals without the need for immune suppression. These studies show that self-renewing cells are abundant in T-ALL and comprise 0.1% to 15.9% of the T-ALL mass. Large-scale single-cell transplantation experiments established that T-ALLs can be initiated from a single cell and that leukemias exhibit wide differences in tumor-initiating potential. T-ALLs also can be introduced into clonal-outcrossed animals, and T-ALLs arising in mixed genetic backgrounds can be transplanted into clonal recipients without the need for major histocompatibility complex matching. Finally, high-throughput imaging methods are described that allow large numbers of fluorescent transgenic animals to be imaged simultaneously, facilitating the rapid screening of engrafted animals. Our experiments highlight the large numbers of zebrafish that can be experimentally assessed by cell transplantation and establish new high-throughput methods to functionally interrogate gene pathways involved in cancer self-renewal.

Self-renewal is a feature of cancer and can be assessed by cell transplantation into immune-compromised or immune-matched animals. However, studies in zebrafish have been severely limited by lack of these reagents. Here, Myc-induced T-cell acute lymphoblastic leukemias (T-ALLs) have been made in syngeneic, clonal zebrafish and can be transplanted into sibling animals without the need for immune suppression. These studies show that self-renewing cells are abundant in T-ALL and comprise 0.1% to 15.9% of the T-ALL mass. Large-scale single-cell transplantation experiments established that T-ALLs can be initiated from a single cell and that leukemias exhibit wide differences in tumor-initiating potential. T-ALLs also can be introduced into clonal-outcrossed animals, and T-ALLs arising in mixed genetic backgrounds can be transplanted into clonal recipients without the need for major histocompatibility complex matching. Finally, high-throughput imaging methods are described that allow large numbers of fluorescent transgenic animals to be imaged simultaneously, facilitating the rapid screening of engrafted animals. Our experiments highlight the large numbers of zebrafish that can be experimentally assessed by cell transplantation and establish new high-throughput methods to functionally interrogate gene pathways involved in cancer self-renewal.

Wang: Pharmacyclics, Janssen: Honoraria, Research Funding.

Introduction

No relevant conflicts of interest to declare.

Abstract 2805

The concept that tumor-initiating cells can co-opt the self-renewal program of endogenous stem cells as a means of enforcing their unlimited proliferative potential is widely accepted, yet identification of specific factors that regulate self-renewal of normal and cancer stem cells remains limited. Using a comparative transcriptomic approach, we identify ZNF521/Zfp521as a conserved hematopoietic stem cell (HSC)–enriched transcription factor in human and murine hematopoiesis whose function in HSC biology remains elusive. Competitive serial transplantation assays using Zfp521-deficient mice revealed that ZFP521 regulates HSC self-renewal and differentiation. In contrast, ectopic expression of ZFP521 in HSCs led to a robust maintenance of progenitor activity in vitro. Transcriptional analysis of human acute myeloid leukemia (AML) patient samples revealed that ZNF521is highly and specifically upregulated in AMLs with MLLtranslocations. Using an MLL-AF9 murine leukemia model and serial transplantation studies, we show that ZFP521 is not required for leukemogenesis, although its absence leads to a significant delay in leukemia onset. Furthermore, knockdown of ZNF521reduced proliferation in human leukemia cell lines possessing MLL-AF9translocations. Taken together, these results identify ZNF521/ZFP521 as a critical regulator of HSC function, which facilitates MLL-AF9–mediated leukemic disease in mice.

The concept that tumor-initiating cells can co-opt the self-renewal program of endogenous stem cells as a means of enforcing their unlimited proliferative potential is widely accepted, yet identification of specific factors that regulate self-renewal of normal and cancer stem cells remains limited. Using a comparative transcriptomic approach, we identify ZNF521/Zfp521 as a conserved hematopoietic stem cell (HSC)–enriched transcription factor in human and murine hematopoiesis whose function in HSC biology remains elusive. Competitive serial transplantation assays using Zfp521-deficient mice revealed that ZFP521 regulates HSC self-renewal and differentiation. In contrast, ectopic expression of ZFP521 in HSCs led to a robust maintenance of progenitor activity in vitro. Transcriptional analysis of human acute myeloid leukemia (AML) patient samples revealed that ZNF521 is highly and specifically upregulated in AMLs with MLL translocations. Using an MLL-AF9 murine leukemia model and serial transplantation studies, we show that ZFP521 is not required for leukemogenesis, although its absence leads to a significant delay in leukemia onset. Furthermore, knockdown of ZNF521 reduced proliferation in human leukemia cell lines possessing MLL-AF9 translocations. Taken together, these results identify ZNF521/ZFP521 as a critical regulator of HSC function, which facilitates MLL-AF9–mediated leukemic disease in mice.

For more than 50 years, investigators have considered a malignant stem cell as the potential origin of and a key therapeutic target for acute myeloid leukemia (AML) and other forms of cancer.1-4The nature and existence of tumor-initiating cells for leukemia and other malignancies have long been the subject of intense and rigorous study; indeed, the promise of the potential to eradicate such cells is clear. However, until recently, deficiencies in our understanding of the nature of these cell populations, coupled with a limited ability to therapeutically exploit their weaknesses, have been limiting factors in realizing the goal of targeting leukemic stem cells (LSCs). Exciting new insights into the fundamental underpinnings of LSCs are now being made in an era in which drug development pipelines offer the potential to specifically target pathways of significance. Therefore, the focus in this new era, characterized by the confluence of understanding LSCs and the ability to target them, is shifting from “if it can be done” to “how it will be done.” Moving from a theoretical stage to this hopeful era of possibilities, new challenges expectedly arise, and our focus now must shift to determining the best strategy by which to target LSCs, with their well-documented heterogeneity and readily evident intra- and interpatient variability. The purpose of this review is therefore both to summarize the key scientific findings pertinent to AML LSC targeting and to consider methods of clinical evaluation that will be most effective for identifying successful LSC-directed therapies.

For more than 50 years, investigators have considered a malignant stem cell as the potential origin of and a key therapeutic target for acute myeloid leukemia (AML) and other forms of cancer.1-4 The nature and existence of tumor-initiating cells for leukemia and other malignancies have long been the subject of intense and rigorous study; indeed, the promise of the potential to eradicate such cells is clear. However, until recently, deficiencies in our understanding of the nature of these cell populations, coupled with a limited ability to therapeutically exploit their weaknesses, have been limiting factors in realizing the goal of targeting leukemic stem cells (LSCs). Exciting new insights into the fundamental underpinnings of LSCs are now being made in an era in which drug development pipelines offer the potential to specifically target pathways of significance. Therefore, the focus in this new era, characterized by the confluence of understanding LSCs and the ability to target them, is shifting from “if it can be done” to “how it will be done.” Moving from a theoretical stage to this hopeful era of possibilities, new challenges expectedly arise, and our focus now must shift to determining the best strategy by which to target LSCs, with their well-documented heterogeneity and readily evident intra- and interpatient variability. The purpose of this review is therefore both to summarize the key scientific findings pertinent to AML LSC targeting and to consider methods of clinical evaluation that will be most effective for identifying successful LSC-directed therapies.

Disease relapse remains a major factor limiting the survival of cancer patients. In the plasma cell malignancy multiple myeloma (MM), nearly all patients ultimately succumb to disease relapse and progression despite new therapies that have improved remission rates. Tumor regrowth indicates that clonogenic growth potential is continually maintained, but the determinants of self-renewal in MM are not well understood. Normal stem cells are regulated by extrinsic niche factors, and the tumor microenvironment (TME) may similarly influence tumor cell clonogenic growth and self-renewal. Growth differentiation factor 15 (GDF15) is aberrantly secreted by bone marrow stromal cells (BMSCs) in MM. We found that GDF15 is produced by BMSCs after direct contact with plasma cells and enhances the tumor-initiating potential and self-renewal of MM cells in a protein kinase B- and SRY (sex-determining region Y)-box–dependent manner. Moreover, GDF15 induces the expansion of MM tumor-initiating cells (TICs), and changes in the serum levels of GDF15 were associated with changes in the frequency of clonogenic MM cells and the progression-free survival of MM patients. These findings demonstrate that GDF15 plays a critical role in mediating the interaction among mature tumor cells, the TME, and TICs, and strategies targeting GDF15 may affect long-term clinical outcomes in MM.

Recent reports have shown that upon expression of appropriate oncogenes, both stem cells and more differentiated progenitor populations can serve as leukemia-initiating cells. These studies suggest that oncogenic mutations subvert normal development and induce reacquisition of stem-like features. However, no study has described how specific mutations influence the ability of differentiating cell subsets to serve as leukemia-initiating cells and if varying such cellular origins confers a functional difference. We have examined the role of the tumor suppressor gene p19ARF in a murine model of acute lymphoblastic leukemia and found that loss of p19ARF changes the spectrum of cells capable of tumor initiation. With intact p19ARF, only hematopoietic stem cells (HSCs) can be directly transformed by BCR/ABL expression. In a p19ARF-null genetic background expression of the BCR/ABL fusion protein renders functionally defined HSCs, common lymphoid progenitors (CLP), and precursor B-lymphocytes competent to generate leukemia stem cells. Furthermore, we show that leukemias arising from p19ARF-null HSC versus pro-B cells differ biologically, including relative response to drug insult. Our observations elucidate a unique mechanism by which heterogeneity arises in tumor populations harboring identical genetic lesions and show that activity of p19ARF profoundly influences the nature of tumor-initiating cells during BCR/ABL-mediated leukemogenesis.

Recent reports have shown that upon expression of appropriate oncogenes, both stem cells and more differentiated progenitor populations can serve as leukemia-initiating cells. These studies suggest that oncogenic mutations subvert normal development and induce reacquisition of stem-like features. However, no study has described how specific mutations influence the ability of differentiating cell subsets to serve as leukemia-initiating cells and if varying such cellular origins confers a functional difference. We have examined the role of the tumor suppressor gene p19ARFin a murine model of acute lymphoblastic leukemia and found that loss of p19ARFchanges the spectrum of cells capable of tumor initiation. With intact p19ARF, only hematopoietic stem cells (HSCs) can be directly transformed by BCR/ABL expression. In a p19ARF-null genetic background expression of the BCR/ABL fusion protein renders functionally defined HSCs, common lymphoid progenitors (CLP), and precursor B-lymphocytes competent to generate leukemia stem cells. Furthermore, we show that leukemias arising from p19ARF-null HSC versus pro-B cells differ biologically, including relative response to drug insult. Our observations elucidate a unique mechanism by which heterogeneity arises in tumor populations harboring identical genetic lesions and show that activity of p19ARFprofoundly influences the nature of tumor-initiating cells during BCR/ABL-mediated leukemogenesis.

Chronic myeloid leukemia (CML) is sustained by a rare population of primitive, quiescent, BCR-ABL+cells and represents an excellent example of a malignancy in which tumor-initiating cells represent the key to disease eradication. CML is also the first malignancy for which targeted therapy has replaced conventional chemotherapy. Within a vast excess of proliferating progenitor cells that express breakpoint cluster region-abelson (BCR-ABL) and are exquisitely sensitive to the tyrosine kinase inhibitor imatinib mesylate (IM) resides a small population of quiescent leukemic cells that, despite higher levels of BCR-ABL transcripts, exhibits innate insensitivity to IM. These cells remain after IM therapy, even when apparently complete responses are achieved, and they probably explain molecular disease persistence. Although it can be argued that patients may survive for many years with low levels of leukemia still present, it is possible to achieve disease clearance at the molecular level following an allogeneic stem cell transplantation. The emergence of drug resistance with IM monotherapy also argues in favor of complete disease eradication that we believe should remain the ultimate therapeutic goal in CML. New approaches to the elimination of these primitive CML cells may thus be crucial to the development of curative strategies.

Chronic myeloid leukemia (CML) is sustained by a rare population of primitive, quiescent, BCR-ABL+ cells and represents an excellent example of a malignancy in which tumor-initiating cells represent the key to disease eradication. CML is also the first malignancy for which targeted therapy has replaced conventional chemotherapy. Within a vast excess of proliferating progenitor cells that express breakpoint cluster region-abelson (BCR-ABL) and are exquisitely sensitive to the tyrosine kinase inhibitor imatinib mesylate (IM) resides a small population of quiescent leukemic cells that, despite higher levels of BCR-ABL transcripts, exhibits innate insensitivity to IM. These cells remain after IM therapy, even when apparently complete responses are achieved, and they probably explain molecular disease persistence. Although it can be argued that patients may survive for many years with low levels of leukemia still present, it is possible to achieve disease clearance at the molecular level following an allogeneic stem cell transplantation. The emergence of drug resistance with IM monotherapy also argues in favor of complete disease eradication that we believe should remain the ultimate therapeutic goal in CML. New approaches to the elimination of these primitive CML cells may thus be crucial to the development of curative strategies.

Introduction: Anti-myeloma BCMA-specific chimeric antigen receptor (CAR) T-cell therapies represent a promising new treatment strategy, with high response rates observed in the early stages of therapy. However, the responses are not durable. One known mechanism of relapse has been traced to the loss of BCMA expression following long-term CAR-T therapy. Another potential reason is that while BCMA CAR-T cells eliminate the bulk of BCMA-positive MM cells, a small subset of BCMA-negative, very drug-resistant MM cells, such as tumor-initiating cells (TICs) survive and seed relapses. There is a strong correlation between the presence of MM TICs with minimal residual disease, acquired drug resistance and relapse. This suggests that TIC-targeted therapies could improve outcomes. We have previously demonstrated that MM cells expressing CD24 also exhibit features of TICs, e.g. self-renewal, increased expression of embryonic stem cell genes and drug resistance. We have generated bispecific CAR-T cells which recognize both BCMA and CD24 antigens and have tested their therapeutic efficacy in MM cells in vitroand in vivomodels.

Background:The outcomes of patients with multiple myeloma (MM) have improved due to treatment advances. However, some patients still experience rapid progression, multiple drug resistance or recurrent relapse. Tumor-initiating cells, also known as cancer stem cells (CSC) in some cancer types, have been speculated to induce recurrence of the disease. The aim of this study is to infer the identity of myeloma-initiating cells (MICs) utilizing single-cell transcriptome analysis and explore the unique biological characteristics relating with high-risk and drug-resistance.

Tumors contain diverse sub-clones, which can lead to tumor persistence and re-growth after therapy. Previous studies suggest that multiple myeloma (MM) sub-clones may contain tumor-initiating cells which maintain growth and proliferation as well as contribute to drug-resistance. Recently, we have confirmed the existence of MM stem cell-like cells by identifying “side population” (SP) cells as an enriched source of tumor-initiating cells with clonogenic and tumorigenic stem cell properties. Targeting this population with novel therapies represents a promising strategy.

Tumors contain diverse sub-clones, which can lead to tumor persistence and re-growth after therapy. Previous studies suggest that multiple myeloma (MM) sub-clones may contain tumor-initiating cells which maintain growth and proliferation as well as contribute to drug-resistance. Recently, we have confirmed the existence of MM stem cell-like cells by identifying “side population” (SP) cells as an enriched source of tumor-initiating cells with clonogenic and tumorigenic stem cell properties. Targeting this population with novel therapies represents a promising strategy.Arsenic trioxide (ATO, As2O3) shows only limited responses compared to anti-MM agents such as bortezomib and lenalidomide in relapsed and refractory MM. Here we evaluated the anti-MM activity of another arsenic compound (arsenic sulfide, As4S4), used in traditional Chinese medicine with greater efficacy and less genotoxicity than ATO, prepared by milling into realgar nanoparticles (NREA). We assessed the effect of NREA on MM stem cell-like SP fraction in the context of the bone marrow stromal cells (BMSCs) to provide the rational for its clinical evaluation.First, we evaluated the effect of both NREA and ATO on MM cell survival. Both drugs significantly decreased survival of 13 MM cell lines in a concentration-dependent manner. Importantly, we observed more potent anti-MM effect with NREA compared to ATO, with IC50 values of NREA 2-4 times lower than ATO. Moreover, we confirmed the effect of NREA on MM cells from patients with primary relapsed/refractory disease: NREA showed a dose-dependent response, with IC50 values in the range 1-2 μM. Significant concentration-dependent increased apoptosis was observed in NREA-treated MM cells. Similarly, decreased mitochondrial membrane potential was more significantly triggered by NREA than ATO. Consequently, NREA modulated the MM cell cycle profile: concentration-dependent treatment induced either G0/G1 (MM.1S and KMS11) or G2/M arrest (RPMI-S and OPM1) at 48h. To compare in vivo anti-tumor activity of NREA and ATO, we used our xenograft murine model of human MM. Tumor-bearing mice were intraperitoneally treated with NREA, ATO or with the respective vehicle. Treatment with NREA triggered more significant tumor growth inhibition compared to ATO at day 8 of treatment.To evaluate the effect of NREA on the SP phenotype of MM cells, we treated MM cells with subtoxic concentrations of NREA and ATO for 72 h. Analysis of MM cells for low intracellular content of Hoechst 33342 (SP fraction) revealed that NREA significantly decreased the percentage of SP cells in MM cells in a dose-dependent manner, whereas increase in SP fraction was observed in ATO-treated cells. To evaluate the effect of the bone marrow microenvironment on targeting the SP cells with NREA, MM cells were cultured alone or with BMSCs, and then analyzed for low intracellular accumulation of Hoechst 33342. Treatment by NREA, but not ATO, significantly depleted SP cells in co-cultures MM cells with BMSCs. Moreover, NREA decreased clonogenic potential, whereas ATO had no effect. Similarly, tumorigenic potential of SP cells was significantly decreased by NREA, but not by ATO, treatment.Overall, our results demonstrate significant anti-tumor activity of NREA against MM cells in vitro. Our in vivo data further show that NREA significantly decreased tumor burden at clinically achievable concentrations of arsenic. Importantly, targeting of SP cells by NREA in the context of BMSCs provides the preclinical rationale for its clinical evaluation.Hideshima: Acetylon Pharmaceuticals: Consultancy. Richardson:Millennium: Membership on an entity’s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

Leukemia is often maintained by a relatively rare subset of tumor-initiating cells, which may be resistant to traditional chemotherapy. Targeting these leukemic stem cells is expected to eliminate or reduce acquired resistance to chemotherapy and disease recurrence, but this goal has been elusive. It's also not clear whether eliminating the 'root' of cancer is sufficient or whether targeting both 'root' and 'branch' (i.e. LSCs and their bulk progeny) is necessary. To test this, we established and characterized a mouse model with clearly defined populations of rare leukemic stem cells (LSCs), abundant leukemic blast cell progeny, and normal hematopoietic stem/progenitor cells (HSPCs). This was achieved by simultaneously activating the Wnt/β-catenin and PI3K/Akt signaling pathways in HSPCs to drive stem cell self-renewal-resulting in successive expansion of HSPCs, LSCs, and leukemic blasts. Functional analysis showed a 1,300-fold increase in leukemia-initiating activity of sorted LSCs compared to bulk blast cells or sorted HSPCs. Since Akt activates β-catenin by phosphorylation at serine 552 and thus represents a molecular link mediating the synergistic, self-renewal promoting activity of these pathways, we sought to specifically target pS552-β-catenin. Unexpectedly, high-throughput screening (HTS) and subsequent validation assays found that doxorubicin (DXR) inhibits pS552-β-catenin with minimal effects on total β-catenin.

Multiple myeloma (MM) pathogenesis has been explained for many years by the cancer biology dogma introduced by Peter Nowell: first, a single plasma cell would be immortalized by an error in the immunoglobulin genes rearrangement process; then, a progressive stepwise acquisition of somatic cell mutations would induce a sequential selection and domination by the fittest clone. In line with this idea of “myeloma stability”, SNP arrays studies in diagnostic-relapse paired samples have revealed the presence of common clonal characteristics. Biologically, the M-protein remains usually constant across MM evolution and further, the variable domain of the rearranged immunoglobulin heavy chain genes (or CDR3 region) has been used as a patient-specific myeloma fingerprint in minimal residual disease (MRD) studies. However, massive genome studies with Next Generation Sequencing (NGS) have challenged this concept, showing a significant intraclonal heterogeneity at diagnosis with the possible presence of several clonal progenitors or tumor-initiating cells.

Stem cells (SCs) have the ability to self-renew long term and differentiate into one or more tissues. Typically, SCs are used sparingly to replenish cells during normal homeostasis. However, even SCs that are quiescent must be able to respond quickly to injury in order to fuel rapid tissue regeneration. How SCs balance self-renewal and differentiation is of fundamental importance to our understanding of normal tissue maintenance and wound repair. Increasing evidence suggests that the regulatory circuitry governing this balancing act is at the root of some types of tumors both in mice and in humans. The skin is an excellent model system to understand how SCs function in normal tissue generation and how this process goes awry in cancer. Many of the principles we have unearthed by studying skin stem cells have parallels to the hematopoietic system. We have identified quiescent and “primed”/active SCs within the skin and shown that primed SCs have a lower threshold for activation to enter tissue regeneration. We have learned that during normal homeostasis, SC behavior is controlled not only through cues received from their microenvironment but also through signals emanating from their differentiating lineages. We have been dissecting how extrinsic signaling to SCs trigger a cascade of transcriptional changes that govern SC activation during tissue development, homeostasis and hair regeneration. Our findings provide new insights into our understanding of the process of SC activation, and in so doing have revealed mechanisms which are also deregulated in a variety of different human cancers. Our goal is to understand how SCs start and stop making tissue, and how this changes in cancer. Our recent discoveries on this topic have led us to the realm of identifying and characterizing cancer SCs (tumor-initiating cells) of squamous cell carcinomas (SCCs) of the skin. We developed a new method to knockdown genes specifically in skin and oral progenitors, enabling us to screen not only the differences between cancer and normal SCs, but also the myriad of gene alterations surfacing from the Human Cancer Sequencing project. Our screens have illuminated new oncogenes and tumor suppressors for SCCs, among the most prevalent and life-threating cancers world-wide that include cancers of lung, esophagus, breast, cervix, prostate, throat and oral tissues. Our findings are unearthing new targets for cancer therapeutics.

We have previously demonstrated that hematological malignancies contain a distinct “side-population” (SP), which is characterized by the active transport of Hoechst fluorescent dye. The mechanisms responsible for Hoechst efflux also contribute to resistance of the SP to chemotherapy. This population has also been shown to be enriched for tumor initiating cells in several human cancer models. We hypothesize that targeting the cancer “SP” cells using immunotherapy may prevent relapse by eliminating the cell population that: is least sensitive to chemotherapy and has tumor repopulating potential. In this study we characterized SP cells in ten human lymphoma cell lines. Five of the 11 lymphoma cell lines tested (HDLM2, L428, Ramos, Toledo, Karpas) had a distinct SP ranging from 0.8–2% of total cells. We subsequently found SP cells with tumor associated markers in biopsy samples from patients with Hodgkin’s Lymphoma, T-cell Lymphoma, and Follicular lymphoma. Culture of the sorted cell lines show that SP but not the non-SP cells produced progeny that were both SP and non-SP cells. Gene expression analysis of the SP showed increased expression of ABCG2 and other ABC transporters that mediate both the transport of Hoechst dye and selected chemotherapeutic agents out of the cell. We evaluated whether lymphoma cell lines with an SP are resistant to the chemotherapeutic drug gemcitabine. The viability of lymphoma lines containing SP cells was significantly higher (mean 59.8%; range 37.4–87.8%%) than the viability of lines without an SP (mean 9.3% range 3.0%–12.2%) when cultured with 10nM gemcitabine for 3 days. The SP component of the lymphoma cells became enriched 10 fold when cultured with gemcitabine. Furthermore, when equal numbers of SP and non-SP cells were grown in separate fractions with gemcitabine, there was reduced viability of the non-SP (2,247 +/−294 mean counts per minute (cpm)) by thymidine assay. In contrast, the viability of the SP was preserved (13,200 +/− 7500 mean cpm). There was no difference in the viability between the untreated SP and Non SP, so that the gemcitabine rather than the Hoechst dye accounts for the differences in viability. Although lymphoma SP cells are resistant to chemotherapeutic agents, they also express higher levels of tumor associated antigens that are known targets for cytotoxic T cells. Hence RT-PCR and immunohistochemistry both show that SP cells are HLA positive and have increased expression of MAGE-4, SSX2, Survivin and NY-ESO transcripts and protein compared to non-SP cells from the same tumor. In IFNγ ELISPOT assays, we have shown that T cells secrete IFNγ in response to SP targets. Hence, lymphoma cell lines and primary lymphoma tissue contain chemotherapy-resistant SP cells which express tumor associated antigens and may be targeted by specific T cells.

We have previously demonstrated that hematological malignancies contain a distinct “side-population” (SP), which is characterized by the active transport of Hoechst fluorescent dye. The mechanisms responsible for Hoechst efflux also contribute to resistance of the SP to chemotherapy. This population has also been shown to be enriched for tumor initiating cells in several human cancer models. We hypothesize that targeting the cancer “SP” cells using immunotherapy may prevent relapse by eliminating the cell population that:

In acute myeloid leukemia (AML), a small fraction of blast cells contains the tumor initiating cells, further referred to as leukemic stem cells (LSCs). LSC resemble hematopoietic stem cells (HSCs) with respect to self renewal capacity and quiescence. Therefore, LSCs are proposed to be therapy resistant. In order to optimally target LSCs and sparing HSC and to monitor therapy, discrimination between LSC is HSC is required. We showed that within the CD34+CD38− stem cell compartment, LSCs can be discriminated from HSC by aberrant expression of markers, including lineage markers like CD7, CD19 and CD56 and the novel LSC marker CLL-1 (van Rhenen et al., Leukemia 2007 and Blood 2007). Too low aberrant marker expression, however, hampers discrimination in part of the cases. Therefore, we investigated additional parameters that would allow to distinguish LSCs from HSCs in CD34 positive AML patients. In 14 out of 48 cases studied, flow cytometry revealed a double population within the CD34+CD38− compartment, characterized by a small but clear difference in forward scatter (FSC, reflecting cell size) and sideward scatter (SSC, reflecting granularity). In 7/14 cases with high marker expression, FSChighSSChigh population coincided completely with the population with aberrant marker expression. In the other cases, marker expression was too moderate to show a complete overlap. The FSClowSSClow population within the CD34+CD38− stem cell compartment is the minor population at diagnosis (median 16%, range 0.2%–92%; n=14), had no expression of aberrant markers and, moreover, closely resembled the FSC/SSC characteristics of normal BM HSCs. In addition, in these patients, the normality of the FSClowSSClow population was also supported by the fact that the CD34 and CD45 antigen density was similar to that of normal BM HSCs. Altogether, this enabled to use FSC/SSC characteristics together with aberrant CD34 and CD45 expression to discriminate between LSC and HSC in cases with low or absent aberrant marker expression (8/48). In addition, the malignant character of the FSChighSSChigh population and the normal character of the FSClowSSClow population could be proven in three AML patients with cytogenetic aberrancies. Patient 1 had a t(8;21) translocation and presented with a CD34+CD38−- population that was CD19 positive (81% of the stem cell compartment) and had FSChighSSChigh properties. FACSsorted cells contained the translocation in 90% of the cells. The CD19 negative population (19% of the stem cell compartment) had FSClowSSClow characteristics and contained 0% t(8;21) cells. In two other AML cases with a cytogenetic aberrancy (t(8;21) and t(15;17), respectively), FSC/SSC characteristics, CD34/CD45 antigen density and aberrant marker expression (CD56 in one case and CLL-1 in the other) were partly overlapping (estimated LSC contribution to the CD34+CD38− compartment was 85% in both cases). Cell sorting on the highest FSC/SSC and marker expression nevertheless resulted in enrichment of cytogenetically aberrant cells (63% and 73%, respectively), while the corresponding FSClowSSClow cells, which missed CD56 and CLL-1 expression, were enriched for cytogenetically normal HSCs (87% and 67%, respectively). The marker and scatter parameters discussed above have generated the possibility to discriminate between LSCs and HSCs and now allows specific detection of LSC in >75 % of the patients. Discrimination between LSCs and HSCs in AML might not only facilitate to establish the therapeutic window of current therapies in terms of LSC specificity, but also allow the identification of new highly AML stem cells specific therapeutic targets. This should ultimately result in more selective therapies, which would be highly effective for AML stem cells, while leaving the normal HSC intact. This work was supported by Netherlands Cancer Foundation KWF.

In acute myeloid leukemia (AML), a small fraction of blast cells contains the tumor initiating cells, further referred to as leukemic stem cells (LSCs). LSC resemble hematopoietic stem cells (HSCs) with respect to self renewal capacity and quiescence. Therefore, LSCs are proposed to be therapy resistant. In order to optimally target LSCs and sparing HSC and to monitor therapy, discrimination between LSC is HSC is required. We showed that within the CD34+CD38− stem cell compartment, LSCs can be discriminated from HSC by aberrant expression of markers, including lineage markers like CD7, CD19 and CD56 and the novel LSC marker CLL-1 (van Rhenen et al., Leukemia 2007 and Blood 2007). Too low aberrant marker expression, however, hampers discrimination in part of the cases. Therefore, we investigated additional parameters that would allow to distinguish LSCs from HSCs in CD34 positive AML patients. In 14 out of 48 cases studied, flow cytometry revealed a double population within the CD34+CD38− compartment, characterized by a small but clear difference in forward scatter (FSC, reflecting cell size) and sideward scatter (SSC, reflecting granularity). In 7/14 cases with high marker expression, FSChighSSChighpopulation coincided completely with the population with aberrant marker expression. In the other cases, marker expression was too moderate to show a complete overlap. The FSClowSSClowpopulation within the CD34+CD38− stem cell compartment is the minor population at diagnosis (median 16%, range 0.2%–92%; n=14), had no expression of aberrant markers and, moreover, closely resembled the FSC/SSC characteristics of normal BM HSCs. In addition, in these patients, the normality of the FSClowSSClowpopulation was also supported by the fact that the CD34 and CD45 antigen density was similar to that of normal BM HSCs. Altogether, this enabled to use FSC/SSC characteristics together with aberrant CD34 and CD45 expression to discriminate between LSC and HSC in cases with low or absent aberrant marker expression (8/48). In addition, the malignant character of the FSChighSSChighpopulation and the normal character of the FSClowSSClowpopulation could be proven in three AML patients with cytogenetic aberrancies. Patient 1 had a t(8;21) translocation and presented with a CD34+CD38−- population that was CD19 positive (81% of the stem cell compartment) and had FSChighSSChighproperties. FACSsorted cells contained the translocation in 90% of the cells. The CD19 negative population (19% of the stem cell compartment) had FSClowSSClowcharacteristics and contained 0% t(8;21) cells. In two other AML cases with a cytogenetic aberrancy (t(8;21) and t(15;17), respectively), FSC/SSC characteristics, CD34/CD45 antigen density and aberrant marker expression (CD56 in one case and CLL-1 in the other) were partly overlapping (estimated LSC contribution to the CD34+CD38− compartment was 85% in both cases). Cell sorting on the highest FSC/SSC and marker expression nevertheless resulted in enrichment of cytogenetically aberrant cells (63% and 73%, respectively), while the corresponding FSClowSSClowcells, which missed CD56 and CLL-1 expression, were enriched for cytogenetically normal HSCs (87% and 67%, respectively). The marker and scatter parameters discussed above have generated the possibility to discriminate between LSCs and HSCs and now allows specific detection of LSC in >75 % of the patients. Discrimination between LSCs and HSCs in AML might not only facilitate to establish the therapeutic window of current therapies in terms of LSC specificity, but also allow the identification of new highly AML stem cells specific therapeutic targets. This should ultimately result in more selective therapies, which would be highly effective for AML stem cells, while leaving the normal HSC intact. This work was supported by Netherlands Cancer Foundation KWF.