CCRL2 affects the sensitivity of myelodysplastic syndrome and secondary acute myeloid leukemia cells to azacitidine.

Better understanding of the biology of resistance to DNA methyltransferase (DNMT) inhibitors is required to identify therapies that can improve their efficacy for patients with high-risk myelodysplastic syndrome (MDS). CCRL2 is an atypical chemokine receptor that is upregulated in CD34+ cells from MDS patients and induces proliferation of MDS and secondary acute myeloid leukemia (sAML) cells. In this study, we evaluated any role that CCRL2 may have in the regulation of pathways associated with poor response or resistance to DNMT inhibitors. We found that CCRL2 knockdown in TF-1 cells downregulated DNA methylation and PRC2 activity pathways and increased DNMT suppression by azacitidine in MDS/sAML cell lines (MDS92, MDS-L and TF-1). Consistently, CCRL2 deletion increased the sensitivity of these cells to azacitidine in vitro and the efficacy of azacitidine in an MDS-L xenograft model. Furthermore, CCRL2 overexpression in MDS-L and TF-1 cells decreased their sensitivity to azacitidine. Finally, CCRL2 levels were higher in CD34+ cells from MDS and MDS/myeloproliferative neoplasm patients with poor response to DNMT inhibitors. In conclusion, we demonstrated that CCRL2 modulates epigenetic regulatory pathways, particularly DNMT levels, and affects the sensitivity of MDS/sAML cells to azacitidine. These results support CCRL2 targeting as having therapeutic potential in MDS/sAML.

Association of acute myeloid leukemia's most immature phenotype with risk groups and outcomes.

The precise phenotype and biology of acute myeloid leukemia stem cells remain controversial, in part because the "gold standard" immunodeficient mouse engraftment assay fails in a significant fraction of patients and identifies multiple cell-types in others. We sought to analyze the clinical utility of a novel assay for putative leukemia stem cells in a large prospective cohort. The leukemic clone's most primitive hematopoietic cellular phenotype was prospectively identified in 109 newly-diagnosed acute myeloid leukemia patients, and analyzed against clinical risk groups and outcomes. Most (80/109) patients harbored CD34(+)CD38(-) leukemia cells. The CD34(+)CD38(-) leukemia cells in 47 of the 80 patients displayed intermediate aldehyde dehydrogenase expression, while normal CD34(+)CD38(-) hematopoietic stem cells expressed high levels of aldehyde dehydrogenase. In the other 33/80 patients, the CD34(+)CD38(-) leukemia cells exhibited high aldehyde dehydrogenase activity, and most (28/33, 85%) harbored poor-risk cytogenetics or FMS-like tyrosine kinase 3 internal tandem translocations. No CD34(+) leukemia cells could be detected in 28/109 patients, including 14/21 patients with nucleophosmin-1 mutations and 6/7 acute promyelocytic leukemia patients. The patients with CD34(+)CD38(-) leukemia cells with high aldehyde dehydrogenase activity manifested a significantly lower complete remission rate, as well as poorer event-free and overall survivals. The leukemic clone's most immature phenotype was heterogeneous with respect to CD34, CD38, and ALDH expression, but correlated with acute myeloid leukemia risk groups and outcomes. The strong clinical correlations suggest that the most immature phenotype detectable in the leukemia might serve as a biomarker for "clinically-relevant" leukemia stem cells. ClinicalTrials.gov: NCT01349972.

Characterization of aldehyde dehydrogenase 1 high ovarian cancer cells: Towards targeted stem cell therapy.

OBJECTIVE: The cancer stem cell (CSC) paradigm hypothesizes that successful clinical eradication of CSCs may lead to durable remission for patients with ovarian cancer. Despite mounting evidence in support of ovarian CSCs, their phenotype and clinical relevance remain unclear. We and others have found high aldehyde dehydrogenase 1 (ALDH(high)) expression in a variety of normal and malignant stem cells, and sought to better characterize ALDH(high) cells in ovarian cancer. METHODS: We compared ALDH(high) to ALDH(low) cells in two ovarian cancer models representing distinct subtypes: FNAR-C1 cells, derived from a spontaneous rat endometrioid carcinoma, and the human SKOV3 cell line (described as both serous and clear cell subtypes). We assessed these populations for stem cell features then analyzed expression by microarray and qPCR. RESULTS: ALDH(high) cells displayed CSC properties, including: smaller size, quiescence, regenerating the phenotypic diversity of the cell lines in vitro, lack of contact inhibition, nonadherent growth, multi-drug resistance, and in vivo tumorigenicity. Microarray and qPCR analysis of the expression of markers reported by others to enrich for ovarian CSCs revealed that ALDH(high) cells of both models showed downregulation of CD24, but inconsistent expression of CD44, KIT and CD133. However, the following druggable targets were consistently expressed in the ALDH(high) cells from both models: mTOR signaling, her-2/neu, CD47 and FGF18/FGFR3. CONCLUSIONS: Based on functional characterization, ALDH(high) ovarian cancer cells represent an ovarian CSC population. Differential gene expression identified druggable targets that have the potential for therapeutic efficacy against ovarian CSCs from multiple subtypes.

Regulation of human hematopoietic stem cell self-renewal by the microenvironment's control of retinoic acid signaling.

The high expression of aldehyde dehydrogenase 1, also known as retinaldehyde dehydrogenase, by hematopoietic stem cells (HSCs) suggests an important role for retinoic acid (RA) signaling in determining the fate of these cells. We found that primitive human bone marrow-derived CD34(+)CD38(-) cells not only highly express aldehyde dehydrogenase 1, but also the RA receptor α. Despite the up-regulation of early components of RA signaling, the downstream pathway remained inactive in the primitive CD34(+)CD38(-) cells. Primitive hematopoietic cells rapidly undergo terminal differentiation when cultured away from their microenvironment; however, we found that inhibition of RA signaling maintained their primitive phenotype and function, and promoted their self-renewal. HSCs reside in a complex microenvironment that enforces the balance between self-renewal and differentiation. The exact physiologic mechanisms by which the niche controls HSC fate remain elusive. The embryonic gonadal microenvironment has recently been shown to determine germ-cell fate by degrading RA through expression of the P450 retinoid-inactivating enzyme CYP26B1. We found that the bone marrow microenvironment similarly can control primitive hematopoietic cell fate via modulation of retinoid bioavailability. Accordingly, we found that bone marrow stromal cell CYP26 was also able to inactivate retinoids in serum, preventing RA signaling. Thus, primitive hematopoietic cells appear to be intrinsically programmed to undergo RA-mediated differentiation unless prevented from doing so by bone marrow niche CYP26. Modulation of RA signaling also holds promise for clinical HSC expansion, a prerequisite for the wide-scale use of these cells in regenerative medicine and gene therapy.

Phase 2 study of rituximab-ABVD in classical Hodgkin lymphoma.

In classical Hodgkin lymphoma, circulating clonotypic malignant cells express CD20, which potentially explains the observed activity of rituximab. This multicenter phase 2 study investigated the combination of rituximab-ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) for stage II-IV untreated classical Hodgkin lymphoma. A goal was to assess the behavior of circulating clonotypic B cells clinically. Of 49 evaluable patients, 69% had stage IIB-IV disease; 8% had CD20(+) Hodgkin and Reed-Sternberg cells. Rituximab-ABVD was generally well tolerated. Delivered relative dose intensity was 94% for AVD and 79% for bleomycin. After 6 cycles, 81% of patients were in complete remission. Only 8% received radiation therapy. The actuarial 3-year event-free and overall survival rates were 83% and 98%, respectively. EBV copy number in plasma fell dramatically during cycle 1 in patients with EBV(+) tumors. Persistence of detectable circulating clonotypic B cells was associated with a greater relapse frequency (P < .05). Rituximab-ABVD and clonotypic B cells warrant additional study in classical Hodgkin lymphoma.

A clinically relevant population of leukemic CD34(+)CD38(-) cells in acute myeloid leukemia.

Relapse of acute myeloid leukemia (AML) is thought to reflect the failure of current therapies to adequately target leukemia stem cells (LSCs), the rare, resistant cells presumed responsible for maintenance of the leukemia and typically enriched in the CD34(+)CD38(-) cell population. Despite the considerable research on LSCs over the past 2 decades, the clinical significance of these cells remains uncertain. However, if clinically relevant, it is expected that LSCs would be enriched in minimal residual disease and predictive of relapse. CD34(+) subpopulations from AML patients were analyzed by flow cytometry throughout treatment. Sorted cell populations were analyzed by fluorescence in situ hybridization for leukemia-specific cytogenetic abnormalities (when present) and by transplantation into immunodeficient mice to determine self-renewal capacity. Intermediate (int) levels of aldehyde dehydrogenase (ALDH) activity reliably distinguished leukemic CD34(+)CD38(-) cells capable of engrafting immunodeficient mice from residual normal hematopoietic stem cells that exhibited relatively higher ALDH activity. Minimal residual disease detected during complete remission was enriched for the CD34(+)CD38(-)ALDH(int) leukemic cells, and the presence of these cells after therapy highly correlated with subsequent clinical relapse. ALDH activity appears to distinguish normal from leukemic CD34(+)CD38(-) cells and identifies those AML cells associated with relapse.

Intraductally administered pegylated liposomal doxorubicin reduces mammary stem cell function in the mammary gland but in the long term, induces malignant tumors.

Previously, we have shown that the intraductal (i.duc) administration of pegylated liposomal doxorubicin (PLD) to Her2/neu transgenic mice is associated with mammary tumor regression and prevention. Exploring the mechanism underlying the protection afforded by PLD, we studied: the effects of i.duc PLD-treatment with a subsequent pregnancy on outgrowth of tumors in Her2/neu mice; whether the i.duc PLD antitumor effect was mediated partially through changes in normal mammary stem cells (MaSCs); and the long-term safety of i.duc PLD into the normal mouse mammary gland. Her2/neu mice were treated with two i.duc injections of PLD given four weeks apart; pregnancy was induced and mice were followed up for changes in physiology, and tumor formation. We found that all pups born to i.duc PLD-treated Her2/neu mice died without weight gain within 7 days after birth. Despite an additional pregnancy, compared to vehicle control PLD-treated Her2/neu mice had a significantly longer latency and lower frequency of tumor development. Mammary epithelial cells isolated from untreated and i.duc PLD-treated 6-8 months-old multiparous FVB/N mice were analyzed for their repopulating ability in mammary fat pads of naïve recipients. Mice were also monitored for abnormalities in mammary gland morphology and function, including tumor formation. PLD-treated FVB/N mice displayed histomorphologic changes and a significant reduction in the outgrowth potential of cells from the mammary glands. Thus, i.duc PLD administration altered the mammary gland structurally and functionally by reducing the MaSC population, which could compromise milk production. Followed long term, i.duc PLD-treated FVB/N mice developed malignant mammary tumors, confirming similar published findings on doxorubicin injected into the mammary gland of rats. Unless there are fundamental species differences in PLD metabolism in rodents and humans, this finding seriously limits the consideration of i.duc PLD use in the clinic for treatment or prevention of breast cancer.

CD34+ cell of origin for immunoglobulin heavy chain variable region unmutated, but not mutated, chronic lymphocytic leukemia.

The clinical course of chronic lymphocytic leukemia (CLL) is highly variable. Immunoglobulin heavy chain variable region (IgHV) mutation status is among the most important prognostic factors, with unmutated IgHV associated with inferior outcomes. CLL presumably arises from mature B cells. However, we hypothesized that IgHV unmutated CLL could arise early in B cell differentiation. We prospectively studied 29 patients with mutated and 88 with unmutated IgHV CLL for the presence of CD34+CD19+ cells harboring CLL chromosomal abnormalities. CD34+CD19+ cells were never detected in mutated CLL. In contrast, a small but distinct population of CD34+CD19+ cells harboring the CLL chromosomal abnormality was present in 86/88 patients with unmutated IgHV across all cytogenetic subtypes. Moreover, the CD34+CD19+ cells generated a 3.8 ± 0.7 fold CLL cell expansion over 3-4 weeks in cultures containing IL-3 and IL-2. Unmutated IgHV CLL appears to arise in CD34+ B cells, which perhaps contributes to its poorer prognosis.

The role of the atypical chemokine receptor CCRL2 in myelodysplastic syndrome and secondary acute myeloid leukemia.

The identification of new pathways supporting the myelodysplastic syndrome (MDS) primitive cells growth is required to develop targeted therapies. Within myeloid malignancies, men have worse outcomes than women, suggesting male sex hormone-driven effects in malignant hematopoiesis. Androgen receptor promotes the expression of five granulocyte colony-stimulating factor receptor-regulated genes. Among them, CCRL2 encodes an atypical chemokine receptor regulating cytokine signaling in granulocytes, but its role in myeloid malignancies is unknown. Our study revealed that CCRL2 is up-regulated in primitive cells from patients with MDS and secondary acute myeloid leukemia (sAML). CCRL2 knockdown suppressed MDS92 and MDS-L cell growth and clonogenicity in vitro and in vivo and decreased JAK2/STAT3/STAT5 phosphorylation. CCRL2 coprecipitated with JAK2 and potentiated JAK2-STAT interaction. Erythroleukemia cells expressing JAK2V617F showed less effect of CCRL2 knockdown, whereas fedratinib potentiated the CCRL2 knockdown effect. Conclusively, our results implicate CCRL2 as an MDS/sAML cell growth mediator, partially through JAK2/STAT signaling.

Circulating clonotypic B cells in classic Hodgkin lymphoma.

Although Hodgkin and Reed-Sternberg (HRS) cells are B lymphoid cells, they are unlike any normal cells of that lineage. Moreover, the limited proliferative potential of HRS cells belies the clinical aggressiveness of Hodgkin lymphoma (HL). More than 20 years ago, the L428 HL cell line was reported to contain a small population of phenotypic B cells that appeared responsible for the continued generation of HRS cells. This observation, however, has never been corroborated, and such clonotypic B cells have never been documented in HL patients. We found that both the L428 and KM-H2 HL cell lines contained rare B-cell subpopulations responsible for the generation and maintenance of the predominant HRS cell population. The B cells within the HL cell lines expressed immunoglobulin light chain, the memory B-cell antigen CD27, and the stem cell marker aldehyde dehydrogenase (ALDH). Clonal CD27(+)ALDH(high) B cells, sharing immunoglobulin gene rearrangements with lymph node HRS cells, were also detected in the blood of most newly diagnosed HL patients regardless of stage. Although the clinical significance of circulating clonotypic B cells in HL remains unclear, these data suggest they may be the initiating cells for HL.

Characterization of chronic myeloid leukemia stem cells.

Although tyrosine kinase inhibitors have redefined the care of chronic myeloid leukemia (CML), these agents have not proved curative, likely due to resistance of the leukemia stem cells (LSC). While a number of potential therapeutic targets have emerged in CML, their expression in the LSC remains largely unknown. We therefore isolated subsets of CD34(+) stem/progenitor cells from normal donors and from patients with chronic phase or blast crisis CML. These cell subsets were then characterized based on ability to engraft immunodeficient mice and expression of candidate therapeutic targets. The CD34(+)CD38(-) CML cell population with high aldehyde dehydrogenase (ALDH) activity was the most enriched for immunodeficient mouse engrafting capacity. The putative targets: PROTEINASE 3, SURVIVIN, and hTERT were expressed only at relatively low levels by the CD34(+)CD38(-)ALDH(high) CML cells, similar to the normal CD34(+)CD38(-)ALDH(high) cells and less than in the total CML CD34(+) cells. In fact, the highest expression of these antigens was in normal, unfractionated CD34(+) cells. In contrast, PRAME and WT1 were more highly expressed by all CML CD34(+) subsets than their normal counterparts. Thus, ALDH activity appears to enrich for CML stem cells, which display an expression profile that is distinct from normal stem/progenitor cells and even the CML progenitors. Indeed, expression of a putative target by the total CD34(+) population in CML does not guarantee expression by the LSC. These expression patterns suggest that PROTEINASE 3, SURVIVIN, and hTERT are not optimal therapeutic targets in CML stem cells; whereas PRAME and WT1 seem promising.

Expression of putative leukemia stem cell targets in genetically-defined acute myeloid leukemia subtypes.

Although most acute myeloid leukemia (AML) patients achieve complete remissions, the majority still eventually relapse and die of their disease. Rare primitive leukemia cells, so-called leukemia stem cells (LSCs), represent one potential type of resistant cell subpopulation responsible for this dissociation between response and cure. Several LSC targets have been described, but there is limited evidence about their relative utility or that targeting any can prevent relapse. LSCs not only appear to be biologically heterogeneous, but the classic immunocompromised mouse transplantation model also has serious shortcomings as an LSC assay. Out data suggest that the most immature cell phenotype that can be identified within a patient's leukemia may be clinically relevant and represent the de facto LSC. Moreover, although phenotypically heterogeneous, these putative LSCs show consistent phenotypes within individual genetically defined groups. Using this LSC definition, we studied several previously described putative LSC targets, CD25, CD26, CD47, CD96, CD123, and CLL-1, and all were expressed across heterogeneous LSC phenotypes. In addition, with the exception of CD47, there was at most low expression of these targets on normal hematopoietic stem cells (HSCs). CD123 and CLL-1 demonstrated the greatest expression differences between putative LSCs and normal HSCs. Importantly, CD123 monoclonal antibodies were cytotoxic in vitro to putative LSCs from all AML subtypes, while showing limited to no toxicity against normal HSCs and hematopoietic progenitors. Since minimal residual disease appears to be a more homogeneous population of cells responsible for relapse, targeting CD123 in this setting may be most effective.

Aldehyde dehydrogenase expression drives human regulatory T cell resistance to posttransplantation cyclophosphamide.

High-dose, posttransplantation cyclophosphamide (PTCy) is an effective strategy for preventing graft-versus-host disease (GVHD) after allogeneic blood or marrow transplantation (alloBMT). However, the mechanisms by which PTCy modulates alloimmune responses are not well understood. We studied early T cell reconstitution in patients undergoing alloBMT with PTCy and the effects of mafosfamide, a cyclophosphamide (Cy) analog, on CD4(+) T cells in allogeneic mixed lymphocyte reactions (MLRs) in vitro. Patients exhibited reductions in naïve, potentially alloreactive conventional CD4(+) T cells with relative preservation of memory CD4(+)Foxp3(+) T cells. In particular, CD4(+)CD45RA(-)Foxp3(+hi) effector regulatory T cells (Tregs) recovered rapidly after alloBMT and, unexpectedly, were present at higher levels in patients with GVHD. CD4(+)Foxp3(+) T cells from patients and from allogeneic MLRs expressed relatively high levels of aldehyde dehydrogenase (ALDH), the major in vivo mechanism of Cy resistance. Treatment of MLR cultures with the ALDH inhibitor diethylaminobenzaldehyde reduced the activation and proliferation of CD4(+) T cells and sensitized Tregs to mafosfamide. Finally, removing Tregs from peripheral blood lymphocyte grafts obviated PTCy's GVHD-protective effect in a xenogeneic transplant model. Together, these findings suggest that Treg resistance to Cy through expression of ALDH may contribute to the clinical activity of PTCy in preventing GVHD.

Novel oligoamine analogues inhibit lysine-specific demethylase 1 and induce reexpression of epigenetically silenced genes.

PURPOSE: Abnormal DNA CpG island hypermethylation and transcriptionally repressive histone modifications are associated with the aberrant silencing of tumor suppressor genes. Lysine methylation is a dynamic, enzymatically controlled process. Lysine-specific demethylase 1 (LSD1) has recently been identified as a histone lysine demethylase. LSD1 specifically catalyzes demethylation of mono- and dimethyl-lysine 4 of histone 3 (H3K4), key positive chromatin marks associated with transcriptional activation. We hypothesized that a novel class of oligoamine analogues would effectively inhibit LSD1 and thus cause the reexpression of aberrantly silenced genes. EXPERIMENTAL DESIGN: Human colorectal cancer cells were treated with the oligoamines and changes in mono- and dimethyl-H3K4 and other chromatin marks were monitored. In addition, treated cells were evaluated for the reexpression of the aberrantly silenced secreted frizzled-related proteins (SFRP) Wnt signaling pathway antagonist genes. Finally, the effects of the LSD1 inhibitors were evaluated in an in vivo xenograft model. RESULTS: Treatment of HCT116 human colon adenocarcinoma cells in vitro resulted in increased H3K4 methylation and reexpression of silenced SFRP genes. This reexpression is also accompanied by a decrease in H3K9me2 repressive mark. Importantly, cotreatment with low doses of oligoamines and a DNA methyltransferase inhibitor highly induces the reexpression of the aberrantly silenced SFRP2 gene and results in significant inhibition of the growth of established tumors in a human colon tumor model in vivo. CONCLUSIONS: The use of LSD1-inhibiting oligoamine analogues in combination with DNA methyltransferase inhibitors represents a highly promising and novel approach for epigenetic therapy of cancer.

Human mast cell progenitors use alpha4-integrin, VCAM-1, and PSGL-1 E-selectin for adhesive interactions with human vascular endothelium under flow conditions.

Mast cells (MCs) are central to asthma and other allergic diseases, and for responses to infection and tissue injuries. MCs arise from committed progenitors (PrMCs) that migrate from the circulation to tissues by incompletely characterized mechanisms, and differentiate in situ in perivascular connective tissues of multiple organs. PrMCs derived in vitro from human cord blood were examined for adhesion molecule expression and their ability to adhere to human umbilical vein endothelial cells (HUVECs) under conditions that mimic physiologic shear flow. The PrMCs expressed alpha(4)beta(1), low levels of beta7, and the beta2-integrins alphaLbeta2 and alphaMbeta2. The PrMCs also expressed PSGL-1, but not L-selectin. At low (0.5 dynes/cm(2)-1.0 dynes/cm(2)) shear stress, PrMCs attached and rolled on recombinant E-selectin and P-selectin and VCAM-1. An anti-PSGL-1 monoclonal antibody (mAb) blocked essentially all adhesion to P-selectin but reduced adhesion to E-selectin by only 40%, suggesting PrMCs express other ligands for E-selectin. PrMCs adhered strongly to tumor necrosis factor-alpha (TNF-alpha)-activated HUVECs, whereas adhesion to interleukin 4 (IL-4)-activated HUVECs was lower. PrMC adhesion to IL-4-activated HUVECs was totally alpha4-integrin- and VCAM-1-dependent. Adhesion to TNF-alpha-activated HUVECs was blocked by 50% by mAbs against alpha4-integrin, vascular cell adhesion molecule-1 (VCAM-1), E-selectin, or PSGL-1, whereas combinations of mAbs to alpha4-integrin plus PSGL-1, or VCAM-1 plus E-selectin, blocked adhesion by greater than 70%. Thus, PrMCs derived in vitro predominantly use alpha4-integrin, VCAM-1, PSGL-1, and other ligands that bind E-selectin for adhesion to cytokine-activated HUVEC monolayers. These observations may explain the abundance of MCs at sites of mucosal inflammation, where VCAM-1 and E-selectin are important inducible receptors.