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The Hedgehog (Hh) pathway has been implicated in a wide variety of human tumors, and early clinical trials with pathway antagonists have validated Hh signaling as a bona fide anticancer target. Despite these encouraging results, several issues surrounding the basic biology of the Hh pathway in human cancers remain unclear. These include the influence of specific oncogenic events on Hh signal transduction, the precise mode of Hh signaling (i.e., autocrine or paracrine) that occurs within human tumors, and the best means to inhibit aberrant pathway activity in the clinical setting. The cancer stem cell (CSC) hypothesis may explain a number of clinical phenomena, such as unchecked self-renewal and the development of metastatic disease, and to some extent, the Hh signaling pathway has been implicated in all of these processes. Therefore, Hh pathway inhibitors may also represent some of the first agents to formally examine the CSC hypothesis in the clinical setting. The diverse nature of Hh signaling in human cancers suggests that disease-specific factors must be carefully considered to identify the optimal use of novel pathway inhibitors. Clin Cancer Res; 16(12); 3130–40. ©2010 AACR.

SummaryTumor-initiating cells (TICs) are defined by their ability to form tumors after xenotransplantation in immunodeficient mice and appear to be relatively rare in most human cancers. Recent data in melanoma indicate that the frequency of TICs increases dramatically via more permissive xenotransplantation conditions, raising the possibility that the true frequency of TICs has been greatly underestimated in most human tumors. We compared the growth of human pancreatic, non-small cell lung, and head and neck carcinomas in NOD/SCID and NSG mice. Although TIC frequency was detected up to 10-fold higher in NSG mice, it remained low (

Severe aplastic anemia (SAA) is a life-threatening bone marrow failure disorder that can be treated with bone marrow transplantation, immunosuppressive therapy, and high-dose cyclophosphamide. Here, we report long-term follow-up on 67 SAA patients (44 treatment-naive and 23 refractory) treated with high-dose cyclophosphamide. At 10 years, the overall actuarial survival was 88%, the response rate was 71% with the majority being complete, and the actuarial event-free survival was 58% in 44 treatment-naive SAA patients. Patients with refractory SAA fared less well after high-dose cyclophosphamide therapy; at 10 years, overall actuarial survival, response, and actuarial event-free survival rates were 62%, 48%, and 27%, respectively. High-dose cyclophosphamide is highly effective therapy for severe aplastic anemia. Large randomized controlled trials will be necessary to establish how results of high-dose cyclophosphamide compare with either bone marrow transplantation or standard immunosuppressive regimens, such as antithymocyte globulin and cyclosporine.

The Hedgehog (Hh) pathway is essential for normal embryonic development and tissue repair. The role of Hh signaling in hematopoiesis has been studied primarily by modulating the activity of Patched and Smoothened, but results have been conflicting. Some studies demonstrate a requirement for pathway activity in hematopoiesis, whereas others report that it is dispensable. Hh activity converges on the Gli transcription factors, but the specific role of these downstream effectors in hematopoiesis has not been reported. We have analyzed hematopoietic stem cell (HSC) and progenitor function in mice with a homozygous deletion of Gli1 (Gli1null). Gli1null mice have more long-term HSCs that are more quiescent and show increased engraftment after transplantation. In contrast, myeloid development is adversely affected with decreased in vitro colony formation, decreased in vivo response to granulocyte colony-stimulating factor (G-CSF), and impaired leukocyte recovery after chemotherapy. Levels of the proto-oncogene Cyclin D1 are reduced in Gli1null mice and may explain the loss of proliferation seen in HSCs and progenitor cells. These data demonstrate that Gli1 regulates normal and stress hematopoiesis. Moreover, they suggest that Gli1 and Smoothened may not be functionally redundant, and direct GLI1 inhibitors may be needed to effectively block HH/GLI1 activity in human disease.

Highly tumorigenic cancer cell (HTC) populations have been identified for a variety of solid tumors and assigned stem cell properties. Strategies for identifying HTCs in solid tumors have been primarily empirical rather than rational, particularly in epithelial tumors, which are responsible for 80% of cancer deaths. We report evidence for a spatially restricted bladder epithelial (urothelial) differentiation program in primary urothelial cancers (UCs) and in UC xenografts. We identified a highly tumorigenic UC cell compartment that resembles benign urothelial stem cells (basal cells), co-expresses the 67-kDa laminin receptor and the basal cell-specific cytokeratin CK17, and lacks the carcinoembryonic antigen family member CEACAM6 (CD66c). This multipotent compartment resides at the tumor-stroma interface, is easily identified on histologic sections, and possesses most, if not all, of the engraftable tumor-forming ability in the parental xenograft. We analyzed differential expression of genes and pathways in basal-like cells versus more differentiated cells. Among these, we found significant enrichment of pathways comprising “hallmarks” of cancer, and pharmacologically targetable signaling pathways, including Janus kinase-signal transducer and activator of transcription, Notch, focal adhesion, mammalian target of rapamycin, epidermal growth factor receptor (erythroblastic leukemia viral oncogene homolog [ErbB]), and wingless-type MMTV integration site family (Wnt). The basal/HTC gene expression signature was essentially invisible within the context of nontumorigenic cell gene expression and overlapped significantly with genes driving progression and death in primary human UC. The spatially restricted epithelial differentiation program described here represents a conceptual advance in understanding cellular heterogeneity of carcinomas and identifies basal-like HTCs as attractive targets for cancer therapy. Disclosure of potential conflicts of interest is found at the end of this article.

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+ALDHhigh 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.

Purpose: Aberrant activation of the Notch signaling pathway is commonly observed in human pancreatic cancer, although the mechanism(s) for this activation has not been elucidated. Experimental Design: A panel of 20 human pancreatic cancer cell lines was profiled for the expression of Notch pathway-related ligands, receptors, and target genes. Disruption of intracellular Notch signaling, either genetically by RNA interference targeting NOTCH1 or pharmacologically by means of the γ-secretase inhibitor GSI-18, was used for assessing requirement of Notch signaling in pancreatic cancer initiation and maintenance. Results: Striking overexpression of Notch ligand transcripts was detectable in the vast majority of pancreatic cancer cell lines, most prominently JAGGED2 (18 of 20 cases, 90%) and DLL4 (10 of 20 cases, 50%). In two cell lines, genomic amplification of the DLL3 locus was observed, mirrored by overexpression of DLL3 transcripts. In contrast, coding region mutations of NOTCH1 or NOTCH2 were not observed. Genetic and pharmacologic inhibition of Notch signaling mitigated anchorage-independent growth in pancreatic cancer cells, confirming that sustained Notch activation is a requirement for pancreatic cancer maintenance. Further, transient pretreatment of pancreatic cancer cells with GSI-18 resulted in depletion in the proportion of tumor-initiating aldehyde dehydrogenase–expressing subpopulation and was associated with inhibition of colony formation in vitro and xenograft engraftment in vivo, underscoring a requirement for the Notch-dependent aldehyde dehydrogenase–expressing cells in pancreatic cancer initiation. Conclusions: Our studies confirm that Notch activation is almost always ligand dependent in pancreatic cancer, and inhibition of Notch signaling is a promising therapeutic strategy in this malignancy.

Folic acid is known to be associated with inflammatory diseases, but the relationship between folic acid and allergic diseases is unclear.The purpose of the study was to examine the relationship between serum folate levels and markers of atopy, wheeze, and asthma.Data were obtained from the 2005-2006 National Health and Nutrition Examination Survey in which serum folate and total IgE levels were measured in 8083 subjects 2 years of age and older. A high total IgE level was defined as greater than 100 kU/L. Allergen-specific IgE levels were measured for a panel of 5 common aeroallergens. Atopy was defined as at least 1 positive allergen-specific IgE level. Doctor-diagnosed asthma and wheeze in the previous 12 months were assessed by means of questionnaire.Serum folate levels were inversely associated with total IgE levels (P.001). The odds of a high total IgE level, atopy, and wheeze decreased across quintiles of serum folate levels, indicating a dose-response relationship between serum folate levels and these outcomes. Each of these associations remained statistically significant after adjusting for age, sex, race/ethnicity, and poverty index ratio. Adjusted odds ratios associated with the fifth quintile of folate relative to the first quintile were as follows: high IgE level, 0.70 (95% CI, 0.53-0.92); atopy, 0.69 (95% CI, 0.57-0.85); and wheeze, 0.60 (95% CI, 0.44-0.82). Higher folate levels were also associated with a lower risk of doctor-diagnosed asthma, but this finding was not statistically significant (odds ratio for fifth quintile vs first quintile, 0.84 [95% CI, 0.70-1.02]).Serum folate levels are inversely associated with high total IgE levels, atopy, and wheeze.

Graft failure after allogeneic blood or marrow transplantation, although generally uncommon, can be a devastating complication. This report includes the outcome of nine patients who received a salvage transplant for failure to engraft after one (n = 8) or 2 (n = 1) prior transplants. Eight patients received allografts from the original donor. All received fludarabine 30 mg/m2 i.v. and alemtuzumab 20 mg i.v. daily from days −6 to −2. Daily CYA was begun on day −2, and the allograft was infused on day 0. The therapy was well tolerated with low toxicity, and all nine patients engrafted, recovering neutrophils at a median of 12 days after transplant. Four patients died: two of relapse, one of a fungal infection in the setting of GVHD and one of multiple sclerosis. The combination of fludarabine and alemtuzumab is an effective and well-tolerated salvage conditioning regimen for patients who experience graft failure after blood or marrow transplants.

Recent evidence suggests that blockade of aberrant Hedgehog signaling can be exploited as a therapeutic strategy for pancreatic cancer. Our previous studies using the prototype Hedgehog small-molecule antagonist cyclopamine had shown the striking inhibition of systemic metastases on Hedgehog blockade in spontaneously metastatic orthotopic xenograft models. Cyclopamine is a natural compound with suboptimal pharmacokinetics, which impedes clinical translation. In the present study, a novel, orally bioavailable small-molecule Hedgehog inhibitor, IPI-269609, was tested using in vitro and in vivo model systems. In vitro treatment of pancreatic cancer cell lines with IPI-269609 resembled effects observed using cyclopamine (i.e., Gli-responsive reporter knockdown, down-regulation of the Hedgehog target genes Gli1 and Ptch, as well as abrogation of cell migration and colony formation in soft agar). Single-agent IPI-269609 profoundly inhibited systemic metastases in orthotopic xenografts established from human pancreatic cancer cell lines, although Hedgehog blockade had minimal effect on primary tumor volume. The only discernible phenotype observed within the treated primary tumor was a significant reduction in the population of aldehyde dehydrogenase–bright cells, which we have previously identified as a clonogenic tumor-initiating population in pancreatic cancer. Selective ex vivo depletion of aldehyde dehydrogenase–bright cells with IPI-269609 was accompanied by significant reduction in tumor engraftment rates in athymic mice. Pharmacologic blockade of aberrant Hedgehog signaling might prove to be an effective therapeutic strategy for inhibition of systemic metastases in pancreatic cancer, likely through targeting subsets of cancer cells with tumor-initiating (“cancer stem cell”) properties. [Mol Cancer Ther 2008;7(9):2725–35]

Multiple myeloma is characterized by the clonal expansion of neoplastic plasma cells within the bone marrow, elevated serum immunoglobulin, and osteolytic bone disease. The disease is highly responsive to a wide variety of anticancer treatments including conventional cytotoxic chemotherapy, corticosteroids, radiation therapy, and a growing number of agents with novel mechanisms of action. However, few if any patients are cured with these modalities and relapse remains a critical issue. A better understanding of clonogenic multiple myleoma cells is essential to ultimately improving long-term outcomes, but the nature of the cells responsible for myeloma regrowth and disease relapse is unclear. We review evidence that functional heterogeneity exists in multiple myeloma and discuss potential strategies and clinical implications of the stem-cell model of cancer in this disease.

Several key observations providing evidence for the cancer stem cell hypothesis and insights into the unique biology of these cells have come from the study of multiple myeloma. These include evidence that cancer cells may be functionally heterogeneous in spite of their genetic homogeneity and that malignant progenitors share many biological features with normal adult stem cells including drug resistance and regulatory processes governing self-renewal. We review studies that have examined clonogenic cells in multiple myeloma, highlight controversies regarding the cell of origin in multiple myeloma, and discuss potential targeting strategies.

Multiple myeloma (MM) is an incurable hematologic malignancy characterized by recurrent chromosomal translocations. Patients with t(4;14)(p16;q32) are the worst prognostic subgroup in MM, although the basis for this poor prognosis is unknown. The t(4;14) is unusual in that it involves 2 potential target genes: fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain (MMSET). MMSET is universally overexpressed in t(4;14) MM, whereas FGFR3 expression is lost in one-third of cases. Nonetheless, the role of MMSET in t(4;14) MM has remained unclear. Here we demonstrate a role for MMSET in t(4;14) MM cells. Down-regulation of MMSET expression in MM cell lines by RNA interference and by selective disruption of the translocated MMSET allele using gene targeting dramatically reduced colony formation in methylcellulose but had only modest effects in liquid culture. In addition, MMSET knockdown led to cell-cycle arrest of adherent MM cells and reduced the ability of MM cells to adhere to extracellular matrix. Finally, MMSET knockdown and knockout reduced tumor formation by MM xenografts. These results provide the first direct evidence that MMSET plays a significant role in t(4;14) MM and suggest that therapies targeting this gene could impact this particular subset of poor-prognosis patients.

An emerging concept in cancer biology is that a rare population of cancer stem cells exists among the heterogeneous cell mass that constitutes a tumor. This concept is best understood in human myeloid leukemia. Normal and malignant hematopoietic stem cell functions are defined by a common set of critical stemness genes that regulate self-renewal and developmental pathways. Several stemness factors, such as Notch or telomerase, show differential activation in normal hematopoietic versus leukemia stem cells. These differences could be exploited therapeutically even with drugs that are already in clinical use for the treatment of leukemia. The translation of novel and existing leukemic stem cell–directed therapies into clinical practice, however, will require changes in clinical trial design and the inclusion of stem cell biomarkers as correlative end points.

In the context of pancreatic cancer, metastasis remains the most critical determinant of resectability, and hence survival. The objective of this study was to determine whether Hedgehog (Hh) signaling plays a role in pancreatic cancer invasion and metastasis because this is likely to have profound clinical implications. In pancreatic cancer cell lines, Hh inhibition with cyclopamine resulted in down-regulation of snail and up-regulation of E-cadherin, consistent with inhibition of epithelial-to-mesenchymal transition, and was mirrored by a striking reduction of in vitro invasive capacity (P < 0.0001). Conversely, Gli1 overexpression in immortalized human pancreatic ductal epithelial cells led to a markedly invasive phenotype (P < 0.0001) and near total down-regulation of E-cadherin. In an orthotopic xenograft model, cyclopamine profoundly inhibited metastatic spread; only one of seven cyclopamine-treated mice developed pulmonary micrometastases versus seven of seven mice with multiple macrometastases in control animals. Combination of gemcitabine and cyclopamine completely abrogated metastases while also significantly reducing the size of “primary” tumors. Gli1 levels were up-regulated in tissue samples of metastatic human pancreatic cancer samples compared with matched primary tumors. Aldehyde dehydrogenase (ALDH) overexpression is characteristic for both hematopoietic progenitors and leukemic stem cells; cyclopamine preferentially reduced “ALDH-high” cells by ∼3-fold (P = 0.048). We confirm pharmacologic Hh pathway inhibition as a valid therapeutic strategy for pancreatic cancer and show for the first time its particular efficacy against metastatic spread. By targeting specific cellular subpopulations likely involved in tumor initiation at metastatic sites, Hh inhibitors may provide a new paradigm for therapy of disseminated malignancies, particularly when used in combination with conventional antimetabolites that reduce “bulk” tumor size. [Cancer Res 2007;67(5):2187–96]

Defective or aberrant differentiation is a hallmark of acute leukemias. This chapter addresses the current strategies being explored in the clinical arena to overcome the “differentiation blockade” by manipulating critical biological and molecular attributes of the leukemic cell. These strategies include the manipulation of cell cycle kinetics, alteration in the balance between pro-survival and pro-death pathways, and chromatin remodeling with resultant modulation of the expression of genes whose products may drive the differentiation process.

Introduction: Despite recent advancements in treatment, pancreatic ductal adenocarcinoma (PDAC) continues to carry a very poor prognosis, which is largely attributable to drug resistance and early metastasis. PDAC cancer stem cells (CSCs) have been shown to play a role in these processes and are associated with worse median survival in patients. In order to better understand the mechanisms that regulate PDAC CSC maintenance and function, we have focused on their interactions with the extracellular matrix (ECM), a major component of the tumor microenvironment in PDAC. Methods: We used PDAC cell lines (Capan-1, MIA PaCa-2, and BxPC-3) and low passage human xenografts to evaluate the effects of a panel of ECM proteins (type I collagen, type IV collagen, fibronectin, and laminin) on CSC function and content. PDAC cells were plated on ECM protein coated plates (Fisher Scientific) and harvested at 72-96 hrs. We plated cells in 3% methylcellulose to assess clonogenic growth and on 8-µm transwell filters (Corning) to assess cell motility. We analyzed ECM-mediated activation of focal adhesion kinase (FAK) by western blot and the expression of phospho-tyrosine-FAK (pY397-FAK) in aldehyde dehydrogenase expressing (ALDH+) CSCs by flow cytometry. We created stable matched PDAC cell lines that overexpress full-length FAK (FAK-FL) or dominant-negative Y397F-FAK protein. We used these cell lines to study in vitro clonogenic growth and motility. We measured in vivo tumor-initiating cell frequency of these cell lines using a limiting dilution assay in NSG mice. We evaluated the efficacy of a small molecule inhibitor of pY397-FAK, PF573228, against PDAC CSCs in vitro. Results: Among the panel of ECM proteins, type I collagen consistently increased in vitro clonogenic growth and motility of all three PDAC cell lines, with a concurrent increase in ALDH+ cells. The effects of type I collagen were specific to ALDH+ cells, since culturing ALDH+ cells on type I collagen led to higher retention of the CSC phenotype compared to culture under normal conditions, whereas culturing ALDHneg cells on type I collagen had no effect. Since ECM proteins can induce FAK activation via Y397-FAK autophosphorylation, we examined the expression of pY397-FAK in ALDH+ cells from cell lines and low-passage xenografts, and found that ALDH+ cells are 6-10-fold enriched in pY397-FAK expression compared to bulk PDAC cells (P Conclusion: Our studies show that interactions with the ECM enhance the number of PDAC CSCs, leading to increased clonogenic growth and cellular motility. We also show that FAK activation is necessary and sufficient for CSC function and may be a target of CSC-directed therapies. Citation Format: Asma Begum, Theodore Ewachiw, Ross McMillan, Vesselin Penchev, Clinton Jung, Ally Huang, Jessica Norberg, NV Rajeshkumar, Anirban Maitra, William Matsui, Zeshaan A. Rasheed. The extracellular matrix and focal adhesion kinase signaling regulate cancer stem cell function in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A70.

e13542 Background: Retinoids are known to cause terminal differentiation and apoptosis in non-APL AML cell lines, but have not proven clinically effective in patients (pts) with MDS or AML. Bexarot...

The Notch signaling pathway is required in both nonneoplastic neural stem cells and embryonal brain tumors, such as medulloblastoma, which are derived from such cells. We investigated the effects of Notch pathway inhibition on medulloblastoma growth using pharmacologic inhibitors of γ-secretase. Notch blockade suppressed expression of the pathway target Hes1 and caused cell cycle exit, apoptosis, and differentiation in medulloblastoma cell lines. Interestingly, viable populations of better-differentiated cells continued to grow when Notch activation was inhibited but were unable to efficiently form soft-agar colonies or tumor xenografts, suggesting that a cell fraction required for tumor propagation had been depleted. It has recently been hypothesized that a small population of stem-like cells within brain tumors is required for the long-term propagation of neoplastic growth and that CD133 expression and Hoechst dye exclusion (side population) can be used to prospectively identify such tumor-forming cells. We found that Notch blockade reduced the CD133-positive cell fraction almost 5-fold and totally abolished the side population, suggesting that the loss of tumor-forming capacity could be due to the depletion of stem-like cells. Notch signaling levels were higher in the stem-like cell fraction, providing a potential mechanism for their increased sensitivity to inhibition of this pathway. We also observed that apoptotic rates following Notch blockade were almost 10-fold higher in primitive nestin-positive cells as compared with nestin-negative ones. Stem-like cells in brain tumors thus seem to be selectively vulnerable to agents inhibiting the Notch pathway. (Cancer Res 2006; 66(15): 7445-52)

The introduction of novel therapeutic agents over the past decade has significantly improved the outcomes of patients with newly diagnosed multiple myeloma (MM). Despite these advances, virtually all patients relapse and develop drug-resistant disease that carries a dismal prognosis. In addition to chemoresistance, relapse and tumor regrowth are dependent on self-renewal to maintain clonogenic growth potential over time. A better understanding of the factors responsible for tumor self-renewal and regrowth may lead to novel therapies and improved survival rates. RAS mutations are common in MM and have been demonstrated to increase in frequency with relapse and disease progression. These findings implicate RAS in chemoresistance, self-renewal, and tumor regrowth upon relapse. We have begun to directly examine the functional role of mutant RAS signaling in MM by stably transducing KRASmut MM cells with a tetracycline-inducible shRNA and found that KRAS knockdown decreases MM clonogenic growth in vitro. Although inhibiting oncogenic RAS may be beneficial for treating relapsed disease it remains an elusive drug target. Recently, our collaborators have developed a novel small peptide RAS signaling inhibitor that mimics the WW domain of IQGAP1 which competes for ERK binding to scaffolds. The WW peptide specifically targets solid tumors containing RAS (or BRAF) mutations and is well tolerated in mouse models. Thus, by targeting only tumorigenic cells it may demonstrate decreased toxicity compared to existing small molecule inhibitors of MAPK signaling. We analyzed gene expression profiling data from the University of Arkansas (GSE2658) and found that elevated IQGAP1 expression levels (top 12.5%) are associated with increased disease-related mortality in MM (log-rank p = 0.0002, HR 4.3, 95% CI 2.0-9.1). This suggests that IQGAP1 may play an important biological role in MM disease progression and that inhibition of IQGAP1 and RAS signaling with the WW peptide may have efficacy and specificity in MM patients. We have treated RAS mutated MM cell lines with the WW peptide or a scrambled (SCR) control peptide and found that the WW peptide effectively decreases pERK levels after 24 hours at doses as low as 20 nM. The WW peptide also decreases cell growth in RAS mutated but not wild-type MM cells by inducing a cell cycle arrest (by BrdU/7-AAD staining). Importantly, treatment of RAS mutated MM cell lines with the WW peptide significantly decreased colony formation in vitro and led to a reduction in the frequency of highly clonogenic CD138neg MM cells. Finally, an immunodeficient mouse xenograft model was used to determine the effect of the WW peptide on engraftment potential. We treated NCI-H929NRAS(G13D) cells with SCR or WW peptide and injected them into NOD/SCID/IL2γchainKO (NSG) mice. Engraftment as detected by kappa light chain ELISA was significantly reduced in the WW peptide treated group. These data further implicate RAS signaling as well as MAPK signaling in MM self-renewal and identify a potential novel therapy for treatment of relapsed/refractory MM. Disclosures No relevant conflicts of interest to declare.

Introduction:Myelodysplastic Syndromes (MDS) represent a heterogeneous group of hematopoietic stem cell (HSC) disorders with varying clinical outcomes, but prognosis uniformly worsens with transformation to secondary acute myeloid leukemia (AML). Despite recent progress in genomics, the mechanisms responsible for disease progression are not fully understood as most of the somatic mutations defined thus far can be found at early stages of the disease. Previous studies have identified aberrant activation of the Hedgehog (Hh) signaling pathway in a subset of AML patients and the expression of the Hh-regulated transcription factor GLI2 correlated with inferior overall and progression free survival. In solid tumors, Hh pathway activation has been associated with metastatic disease progression, and we examined its role in MDS progression and transformation to AML. Methods/Results: We initially quantified changes in Hh pathway activity in CD34+ cells isolated from serial bone marrow samples collected from MDS patients at the time of diagnosis and following progression to AML. We found that the expression of the Hh target genes GLI1 and PTCH1 was increased in 67% of patients (4/6) suggesting that pathway activation was involved in the development of secondary AML. We also analyzed gene expression in 135 MDS patients and found significantly higher GLI2 expression in high-risk MDS (N=80) compared to low-risk MDS (N=55) (p=0.036). In addition, bone marrow blast percentage was significantly higher in the MDS cohort with higher GLI2 expression (mean±SEM=7.1±0.7%) than with lower expression (5.4±0.5%, p=0.039). In order to mechanistically study the effects of Hh pathway activation on MDS progression, we studied mice expressing the Nup98-HoxD13 (NHD13) fusion gene under the control of the vav promoter that generates progressive cytopenia and, in some animals, progression to AML. We crossed NHD13 mice with mice conditionally expressing the constitutively active mutant of the Hh signal transduction regulator Smoothened (SmoM2) in hematopoietic cells expressing Mx1-Cre. The survival of double transgenic animals (NHD13/SmoM2) was significantly shorter compared to mice expressing NHD13 (median survival of 3 months vs. 12 months, p Within the bone marrow, hematopoietic stem/progenitor cells (Lin-Sca1+cKit+) were depleted within both NHD13 and NHD13/SmoM2 mice. To examine the impact of Hh activation on clonogenic growth and self-renewal potential, we plated bone marrow cells in methylcellulose and found that NHD13/SmoM2 cells contained a significantly higher number of CFU compared to NHD13 cells (37 vs. 2, p=0.002) that was sustained with subsequent passages (400 vs. 30, p Conclusions:We found that the Hh signaling pathway may be aberrantly activated in a subset of secondary AML patients progressing from MDS. We also demonstrated that activation of Hh signaling induces fatal AML in a mouse model of MDS characterized by inferior survival and widespread expansion of immature myeloid cells. This disease progression is driven by the acquisition of self-renewal and tumor initiating potential in differentiated Lin+ hematopoietic cells. Our findings suggest that Hh pathway inhibition may be a promising approach for AML arising from MDS. Figure 1 Figure 1. Disclosures Maciejewski: Alexion: Speakers Bureau; Celgene: Speakers Bureau.

Multiple myeloma (MM) remains an incurable malignancy, at least in part, due to minimal residual disease (MRD). It has been hypothesized that MRD is influenced by the survival promoting aspects of the bone marrow tumor microenvironment (TME) and populations of putative MM cancer stem cells. Therefore, identification of survival determinants specific to the TME and putative MM stem cells is critical for the rational design of therapy and elimination of MRD1. Within the context of the TME, we have shown that collaborative signaling between β1 integrin-mediated adhesion to fibronectin (FN) and Interleukin-6 (IL-6) confers a more malignant phenotype via amplification of STAT3 activation2. We have validated the FN-adhesion-dependent amplification of IL-6-induced STAT3 phosphorylation in MM patient specimens by flow cytometry. Further characterization of the events modulated under these culture conditions with quantitative phospho-tyrosine profiling identified 193 differentially phosphorylated peptides after adhesion to FN, treatment with IL-6, and the combination. Phosphopeptides represented proteins involved in signal transduction, cytoskeleton assembly/adhesion, transcription, RNA processing, metabolism, and the cell cycle. Seventy-seven phosphorylations were up-regulated upon adhesion, including PYK2/FAK2, paxillin, CASL, and p130CAS consistent with focal adhesion (FA) formation. Western blot analysis demonstrated PYK2 autophosphorylation of Y402 following myeloma cell adhesion to FN or bone marrow stromal cells (BMSC), correlating with the amplification of IL-6-induced STAT3 and JAK1. We hypothesized that the collaborative signaling between β1 integrin and the IL-6 signal transducer, gp130, was mediated by FA formation and PYK2. Although PYK2 has been linked to downstream signaling events of other JAK-dependent cytokine receptors, it has not been associated with a STAT3 amplification mechanism within the Type I or Type II cytokine receptor families3-6. Assessing downstream gene expression, we also demonstrated that adhesion to FN alone or co-stimulation with FN and IL-6 upregulated mRNA and protein expression of the MM survival-associated gene, deptor7, and this enhanced expression was abrogated by RNAi knockdown of STAT3 or PYK2. Both pharmacological and molecular targeting of PYK2 attenuated the amplification of STAT3 phosphorylation in adhered cells. Importantly, MM cells co-cultured with patient BMSCs showed similar gp130 and β1 integrin-specific enhancement of PYK2, JAK1, STAT3 signaling when adhered to patient BMSCs, demonstrating that PYK2 modulates co-stimulation specific STAT3 signaling in more complex, clinically relevant models of the microenvironment. Interestingly, targeting PYK2 with antisense oligonucleotides (ISIS Pharmaceuticals) or with the dual PYK2/FAK (Focal adhesion kinase) tyrosine kinase inhibitors VS-4718, VS-6062, and VS-6063 (Verastem, Inc.) induced preferential MM cell killing in the context of adhesion to patient BMSCs, but not in monoculture or under transwell co-culture conditions. We confirmed that this cell death correlated with inhibition of BMSC-induced PYK2 and STAT3 phosphorylation. Further analysis similarly demonstrated a preferential reduction in colony formation in BMSC-adherent MM cell lines and patient specimens treated with VS-6063 relative to MM cells in the absence of BMSCs. Lastly, we examined the effects of targeting PYK2 with VS-6063 in ALDH+ MM cancer stem cells. We demonstrated that co-culture with BMSCs increased the percentage of ALDH+ MM cancer stems relative to monoculture. VS-6063 decreased the percentage of the ALDH+ cells associated with BMSC to greater degree than MM cells without BMSC. These data identify a novel PYK2-mediated survival pathway activated within the context of multiple microenvironmental cues, suggesting that PYK2 is a putative TME-specific therapeutic target. Further, putative MM cancer stem cells are sensitive to PYK2 inhibition by the FAK/PYK2 inhibitor VS-6063. As such, these data indicate that PYK2 may be critical target for the eradication of MRD by targeting both MM cells in the context of TME and MM cancer stem cells. These results suggest that MM represents a compelling clinical development path for FAK/PYK2 inhibitors, such as VS-4718 and VS-6063, in clinical trials focusing on eradication of MRD. Disclosures Shain: Onyx/Amgen: Research Funding; Celgene: Research Funding; Envision/Celgene: Speakers Bureau; L&M Healthcare/Onyx/Amgen: Speakers Bureau. Ring:Verastem: Employment. Pachter:Verastem Inc.: Employment, Equity Ownership.

We retrospectively analyzed 83 consecutive recipients of donor lymphocyte infusions (DLI) after allogeneic transplantation for factors associated with disease response and graft-versus-host disease (GVHD). DLI was highly effective in relapsed chronic phase chronic myeloid leukemia (CML), with 71% of patients achieving durable complete remissions (CR). In relapsed acute myeloid leukemia, DLI led to durable CRs in 31% of patients; the rate was

Rituximab, an anti-CD20 monoclonal antibody used to treat B cell lymphoproliferative disorders and autoimmune diseases, kills cells through complement dependent cytotoxicity, antibody-dependent cellular toxicity and apoptosis. A mechanism of resistance to rituximab is upregulation of the complement regulatory proteins, CD59 and CD55. Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic disorder caused by PIGA mutations that lead to a loss of all glycosylphospatidylinositol (GPI)-anchored proteins including, CD55 and CD59. We compared the cytotoxic activity of rituximab against a PNH B cell line, LD -, and the isogenic cell line LD - PIGA + in which GPI-anchor expression was restored by stable transfection of PIGA. The PNH cell line was more sensitive to rituximab-mediated killing than the LD - PIGA + cells. Biochemical disruption of GPI anchors with phosphatidylinositol specific phospholipase C (PIPLC), a phospholipase that cleaves GPI-anchored proteins, also increased rituximab-mediated killing. Thus, genetic and biochemical interruption of GPI anchor proteins augments sensitivity to rituximab.

Interferon-alpha (IFN-alpha) is a pleotropic cytokine that has clinical activity against a wide variety of malignancies, including multiple myeloma (MM). In vitro, IFN-alpha has diverse effects on both normal and malignant cells, however, the exact mechanisms responsible for its clinical anti-tumour activity remain unclear. We found that IFN-alpha inhibited MM cell proliferation in association with cell cycle arrest at G1 and limited the clonogenic growth of both MM cell lines and primary patient specimens. At the doses tested, IFN-alpha was not cytotoxic, but induced terminal plasma cell differentiation resulting in the loss of clonogenicity. These activities were markedly enhanced by the major MM growth factor interleukin 6 (IL-6). Moreover, IL-6 was required for this process, as neutralizing antibodies against IL-6 inhibited the effects of IFN-alpha. IL-6 also induced MM cell terminal differentiation when combined with a second, unrelated, antiproliferative agent bryostatin-1, suggesting that its differentiating activities are preferentially enhanced in the presence of agents that inhibit cell cycling. These results suggest that the differentiating activities of IFN-alpha may play a role in its clinical antimyeloma activity and provide the rationale for clinical differentiation therapy in MM.

262 Background: We and others have identified aldehyde dehydrogenase (ALDH) activity as a marker of pancreatic cancer stem cells (or tumor-initiating cells). The presence of cancer stem cells (CSCs) has been associated with decreased survival and treatment resistance in pancreatic adenocarcinoma. We investigate the role of ALDH expression in predicting survival and patterns of disease recurrence in patients treated with chemoradiation (CRT) following pancreatectomy. Methods: Tissue microarrays using surgical specimens from 1998 to 2002 were stained for ALDH1 and scored as ALDH-positive or ALDH-negative by two expert pancreatic pathologists blinded to patient outcomes. Physician documentation and radiology reports were used to determine follow-up information. Time to local failure (TLF), time to distant metastases (TDM), progression-free survival (PFS), and overall survival (OS) were analyzed using SPSS software. Results: Previously we found that ALDH expression was associated with worse OS in a cohort of 269 patients with resected pancreatic adenocarcinoma (Rasheed, JNCI 2009). From this cohort, adjuvant treatment information was available for 87 patients with ALDH-negative tumors (48.6%) and 41 patients with ALDH-positive tumors (45.6%). In patients treated with adjuvant CRT, median overall survival was superior in the ALDH-negative cohort vs. the ALDH-positive cohort, 26.3 months vs. 18.2 months (p=0.011). Further, in patients treated with adjuvant CRT, ALDH-negative patients had statistically greater TLF, TDM, and PFS than their ALDH-positive counterparts (see table). On multivariate analysis, ALDH positive tumor staining (HR 1.94, p=0.004) and tumor grade (HR 1.54, p=0.041) predicted lower OS, and ALDH positive tumor staining (HR 1.83, p=0.008), tumor grade (HR 1.52, p=0.038), and tumor size >3 cm (HR 1.65, p=0.023) predicted decreased PFS. Conclusions: This study suggests that adjuvant CRT improves TLF, TDM, PFS, and OS in patients with localized pancreatic adenocarcinoma not enriched with ALDH-expressing CSCs. Laboratory studies will help elucidate the mechanisms of treatment resistance in ALDH expressing CSCs.

Background AlloBMT is a potentially curative treatment for multiple myeloma (MM). However, its effectiveness has been compromised by high transplant related mortality (TRM) and acute and/or chronic graft-versus host disease (GvHD). Our development of PTCy has reduced the incidence of GvHD allowing safe and effective related haploidentical alloBMT. PTCy promotes tolerance in both alloreactive host and donor T cells, leading to suppression of both graft rejection and GVHD after alloBMT. Patients and Methods As part of an IRB-approved study we performed a retrospective analysis on all patients with MM who underwent alloBMT followed by PTCy. A total of 39 patients who were transplanted between 2003 and 2011 were identified. All patients received 2 doses of PTCy 50mg/kg on days 3 and 4 after alloBMT. High risk MM was defined as having any one of the following: del(13q) by cytogenetics or t(4;14), t(14;16), t(14;20), -17p ,+1q21 on FISH/cytogenetics. International Staging System (ISS) stage was based on beta2 microglobulin and albumin at diagnosis of symptomatic MM. The definitions of progression, stable disease and response were as per the International Myeloma Working Group criteria. Survival probability was determined using Kaplan-Meier method and differences assessed with the log-rank test. Cox regression was used to model prognostic factors with respect to progression-free (PFS) and overall survival (OS) rates. Results The median follow-up for living patients was 80 months (22-115). The minimum follow up was 23 months. The median age at BMT was 54 (range 37-70). 30 patients (77%) received a transplant from a matched sibling donor. Of the remaining 9 patients, 7 (18%) received a transplant from a haploidentical donor and 2 (5%) from a matched unrelated donor. 28 patients (72%) were in > PR at the time of alloBMT. Only 1 patient (3%) was in a CR prior to alloBMT. Thirty patients (77%) received reduced intensity conditioning and the remaining 9 patients (23%) received myeloablative conditioning. 28 patients (72%) received unmanipulated bone marrow, while remaining 11 patients (28%) received mobilized peripheral blood cells. The median time between diagnosis and alloBMT was 10.6 months (4.1-178.7). Cytogenetics and FISH for evaluating high risk MM were available for 26 patients (66.6%) and of those 15 patients (58%) had high risk features. 28 patients (72%) were evaluable for ISS at diagnosis and of those 13 patients (46%) had ISS III at diagnosis. 36 patients (92%) had been treated with either bortezomib based or immunomodulatory (Imid) based therapy prior to alloBMT. 7 patients (18%) had received an autologous transplant prior to alloBMT. 20 patients (51%) are alive after a median follow up of > 6 years after alloBMT. Only 1 (2.5%) patient died from complications related to alloBMT. At last follow up, 6 patients (15%) are in sustained first complete remission after alloBMT. Of the 9 patients who received myeloablative prep, 6 are alive and 3 are in sustained CR. At 6 months following alloBMT, 12 patients (31%) were in a deeper response compared to their pre-transplant status. 15 patients (38%) developed > grade 2 acute GvHD at a median of 1.5 (range 0.6-4.5) months. Only 3 patients (8%) developed grade 3 acute GvHD and there was no grade 4 acute GVHD. 14 (36%) patients received systemic treatment for acute GvHD. 5 (13%) patients developed chronic GvHD. The median OS was 96 months, and the median PFS was 14 months (95% CI 6.2-32.8). Chronic GvHD was significantly associated with PFS, with a median PFS of 90 months in patients who developed cGvHD compared to 11 months in patients who did not (hazard ratio = 0.3, 95% CI 0.07-1.25). The major cause of death was disease progression. Conclusion The use of PTCy led to a very low TRM in MM, including with related haploidentical donors. Although only a minority of patients maintained long-term PFS, the prolonged OS allows incorporation of novel post-alloBMT strategies to improve disease control. Disclosures: Off Label Use: Outcomes of Allogeneic Blood Or Marrow Transplantation (AlloBMT) In Multiple Myeloma With Post-Transplantation Cyclophosphamide (PTCy).

Background Prevnar, is a multi-valent conjugate vaccine given to children and adults over 50 for the prevention of Streptococcus pneumonia, otis media and pneumococcal pneumonia. The conjugate in Prevnar is a CRM-197 protein molecule which is a nontoxic recombinant Diphtheria toxin. Prevnar serves as an excellent tool in monitoring overall immune response changes in myeloma patients’ pre and post treatment. Humoral B-cell responses can be measured by antibody responses to the pneumococcal antigens, while T cell responses to CRM-197. Clinical Study We previously conducted a study to determine the efficacy of lenalidomide to augment vaccine specific responses in patients with myeloma. Two cohorts of patients were studied. In cohort A (N=10), the first Prevnar vaccine was given two weeks prior to starting lenalidomide and the second vaccine on day 14 of cycle 2 of lenalidomide. In cohort B (N=7), both Prevnar vaccines were given on lenalidomide (day 14 of cycle 2 and 4). As we previously reported patients in cohort B had an overall better B and T cell response to Prevnar compared to cohort A. These responses were due to an overall change in B and T cell phenotype attained with lenalidomide therapy. Results Prospectively, patients in cohort B also had an unexpected overall increase in disease response and in response duration. In Cohort A only 10% of patients responded to therapy while 60% of patients in Cohort B had a clinical response. The patients with a measurable clinical response had a 5-fold increase in the percentage of tumor specific bone marrow (BM) T cells after two vaccinations with Prevnar whereas the non-responding patients had no increase in tumor specific BM T cells. Parelleling the anti-tumor response, responders showed a 15 fold increase in CRM-197 specific BM T cells after the second vaccination. Patients with no clinical response showed minimal CRM-197 T cell immunity. CRM-197 is a specific inhibitor of HB-EGF; syndecan-1 (CD138) is an HB-EGF co-receptor as well as a marker for myeloma plasma cells. We hypothesized that HB-EGF specific responses produced by vaccination with the Prevnar vaccine, and CRM-197 specifically, may have contributed to the overall increased clinical responses in our clinical trial. Responding patients had a 5-fold increase in HB-EGF specific BM T cells after vaccine 2 while clinical non-responders had no increase in HB-EGF specific BM T cells. T cells specificity for purified HB-EGF correlated with both CRM-197 and tumor specific responses. Finally the myeloma cell lines U266, H929, KMS-11 and KMS-12 co-stained for CD138 and HB-EGF with 47% of CD138+ myeloma cells co-expressing HB-EGF. Conclusions We hypothesize that the CRM-197 moiety of the Prevnar vaccine can prime T cell responses against HB-EGF on plasma cells. This immune response, in turn, weakens the tumor stromal interactions in the tumor microenvironment and potentially enhances the anti-tumor efficacy of immunomodulatory drugs such as lenalidomide. Therefore, Prevnar may possibly serve as a candidate anti-myeloma vaccine. Disclosures: No relevant conflicts of interest to declare.

Epstein-Barr virus (EBV) associated cancers have traditionally been thought to harbor the viral genomes as nuclear plasmids in all or nearly all of a patient's tumor cells as determined by in situ hybridization (ISH). We discovered EBV in 4 out of 7 standard multiple myeloma (MM) cell lines. In these positive cell lines, EBV was found in only a subpopulation of cells. For example, in MM.1S (a MM cell line) less than 1% of the cells harbored viral DNA as determined by limiting dilution PCR. These rare infected cells carried approximately 20 EBV copies. Fluorescence in situ hybridization (FISH) confirmed the presence of multiple copies of the EBV genome in rare cells. Immunofluorescence similarly detected the presence of viral genomes expressing EBV nuclear antigen (EBNA) - 1 and 2 in rare cells. Reverse transcriptase PCR confirmed EBNA1 and EBNA2 viral RNA expression. The cells that harbor virus are phenotypically distinct CD19+CD138− B cells. The phenotype overlaps with that identified as a MM cancer stem cell population. Viral genomes were not detected in the plasma cell-like population (CD19−CD138+). The results are consistent with the possibility that EBV is present in MM cancer stem cells in some MM cell lines and that virus is lost as these MM cancer stem cells with a B cell-like phenotype differentiate into plasma cells. Introduction of a dominant negative shRNA of the EBV EBNA-1, a protein essential for viral replication, decreased the proliferation of only the MM cell lines which were positive for EBV. Insertion of the dominant negative EBNA-1 shRNA had no effect on EBV negative MM cell lines, suggesting that EBV plays a role in the growth kinetics of cell lines even when present in a tiny percentage of cells. These results suggest that in some MM lines, EBV is harbored in a B cell-like progenitor population that overlaps with the putative cancer stem cell fraction. The loss of EBV inhibits cell proliferation and that EBV is lost during the B-cell to plasma cell differentiation process. The results presented here suggest that identifying the EBV status of MM patients prior to treatment might lead to a more efficient course of therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A50. Citation Format: Sunetra Biswas, Meir Shamay, Courtney M. Shirley, Raluca Yonescu, William H. Matsui, Ivan Borrello, Richard J. Jones, Richard F. Ambinder. Epstein-Barr virus in the putative stem cell fraction of some multiple myeloma cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A50.

In his letter to the editor, Dr Kuppers calls attention to additional support that clonotypic B cells are present in Hodgkin lymphoma (HL) and may represent HL stem cells. We were unaware of Jansen and colleagues' prior work describing such cells in HL lymph nodes,[1][1],[2][2] and we thank Dr Ku

The role of multiple developmental signaling pathways in the regulation of hematopoiesis has been controversial. In this issue of Cell Stem Cell, two separate reports (Hofmann et al., 2009; Gao et al., 2009) examine whether the Hedgehog signaling pathway modulates normal adult hematopoietic stem cells and blood formation.

Reply to ‘Quantification of marrow CD34+ cells: an important tool for distinguishing between hypoplastic myelodysplastic syndromes and aplastic anemia’ by A Orazi

Pancreatic ductal adenocarcinoma (PDAC) is highly lethal with a survival rate of less than 6 months for the majority of patients. In order to better understand its development we hypothesized that there is cellular heterogeneity in murine and human PDAC precursor lesions (PanINs). We utilized two independent models of murine PanIN (mPanIN) formation to drive oncogenic KrasG12D expression in the murine pancreas during development (KCPdx1) or specifically in adult acinar cells (KCiMist1). The mice were crossed to the mTmG reporter mouse to map the fate of cells expressing Kras (KCiMist1G) that are marked by expression of GFP. We used transmission electron microscopy on mPanIN-containing tissue to identify a morphologically distinct rare cell type present in both models of mPanIN. These cells morphologically resemble tuft cells, which are characterized by their brush-like morphology, including apically projecting microvillae and the expression of DCLK1 and AcTub. Using Flourescence Activated Cell Sorting Techniques (FACS), we were able to isolate DCLK1HI/GFP+ murine PanIN tuft cells expressed in the KCiMist1G model. We discovered that these cells have an increased ability to form PanIN spheres in vitro relative to GFP+ cells alone, indicative of a progenitor PanIN stem cell function. Using immunofluorescent labeling on a human PanIN (n=23) and metastatic PDAC (n=10) tissue arrays, we determined that in 15% of PanIN lesions, 13-30% of the cells have detectable levels of AcTub, with a primarily apical pattern of labeling. In contrast, over 80% of metastatic PDAC cells are AcTub+, but they no longer exhibit an apical pattern of labeling. Applying our cell surface FACS strategy to four different human pancreatic cancer cell lines and three early passage human xenografts, we determined that 2-5% of human pancreatic cancer cells have detectable levels of cell-surface AcTub and DCLK1. We have functionally analyzed the tumor-initiating capacity of cell surface AcTubHI cells using in vitro tumor sphere experiments. In this assay, cell surface AcTubHI cells show a statistically significant 100-fold increase in tumor sphere-forming capacity relative to cell surface AcTubLOW cells. We further evaluated the functional significance of cell surface AcTub by analyzing the in vivo tumor initiating capacities of cell surface AcTubHI and AcTubLOW cells from a human PDAC xenograft. Cell surface AcTubHI cells exhibited significantly increased tumor initiating frequency relative to the AcTubLOW population (**p Citation Format: Jennifer M. Bailey, Janivette Alsina, Zeshaan Rasheed, Ya-Yuan Fu, Florencia McAllister, Pankaj Pasricha, William Matsui, Anirban Maitra, Steven Leach. A morphologically distinct cancer initiating cell in human and murine PanIN and pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5008. doi:10.1158/1538-7445.AM2013-5008

Abstract 4898 Despite improved response rates with the use of novel agents, the vast majority of patients with multiple myeloma (MM) experience disease relapse and progression. This clinical pattern suggests the cells responsible for tumor regrowth persist following treatment, and we previously demonstrated that highly tumorigenic and self-renewing MM cells are relatively resistant to a wide range of anti-myeloma agents. These MM CSCs are present in the peripheral blood circulation and express memory B cell surface antigens (CD19 and CD27) and the stem cell marker aldehyde dehydrogenase (ALDH). We have also studied cellular mechanisms regulating self-renewal and found that MM CSCs express increased levels of telomerase activity (TA). In preclinical models, the novel telomerase inhibitor imetelstat has been found to inhibit CSCs from a wide range of tumor types, and we reported that imetelstat reduced TA in MM CSCs resulting in the loss of self-renewal and clonogenic growth potential. In an open label phase II clinical trial initiated in February 2011, previously treated MM patients (pts) (n=6) with stable but detectable disease following treatment with an iMid or proteasome inhibitor received single agent imetelstat (7.5–9.4 mg/kg IV on days 1 and 8 every 21–28 days). Additional pts (n=4) receiving lenalidomide maintenance and whose disease was stable for at least three months have also been enrolled. Pts received imetelstat therapy until evidence of disease progression or toxicity. Median age is 62.5 years (range 53–68); median number of prior therapies is 1 (range 1–4) including 4 pts with prior autologous transplant and 1 pt with prior mini-allogeneic transplant; 8 pts with prior bortezomib use; 9 pts with prior iMiD use; and 4 pts with concurrent lenalidomide use with receipt of lenalidomide for a median of 11.38 months (range 4.51–19.21 months) prior to initiation of imetelstat. As of July 30, 2012, 6 pts remain on study. Four pts were discontinued from imetelstat therapy after receiving a median of 7 doses of imetelstat either due to disease progression (n=2) or hematologic toxicity (n=2). Cytopenias were the most frequently reported toxicity with 8 of 10 pts demonstrating grade 3–4 thrombocytopenia and neutropenia during cycle 2, often requiring dose reductions or holds in subsequent cycles. The frequency of circulating MM CSCs (CD19+CD27+ALDH+) was quantified by flow cytometry. Circulating MM CSCs could be detected prior to the initiation of imetelstat (mean 10.7 × 10e3 cells/ml, range 17–53 × 10e3) in 8 of the 9 pts who have completed at least 2 cycles of treatment with imetelstat. In the single remaining patient, circulating CSCs were assessed from Cycle 2 onwards. Over the course of treatment, the frequency of MM CSCs decreased significantly, on average 2 fold every 30 days (Fig 1), in 8 of the 9 patients studied despite no upgrades in clinical response as per IWG criteria. In two pts who received 4 and 6 cycles of single agent imetelstat respectively, standard responses detected as decreasing or plateaued serum M protein or light chain levels were sustained over 4 months following discontinuation (Fig 2). Moreover, these delayed responses occurred in the absence of any additional therapy. These findings demonstrate that imetelstat rapidly decreases circulating MM CSCs. In addition, several patients experienced delayed, but sustained, clinical responses as measured by standard criteria. Therefore, imetelstat may have therapeutic implications for MM and other malignancies driven by CSCs. Figure 1. Serial measurement of CSCs in patients (n=9) treated with imetelstat Figure 1. Serial measurement of CSCs in patients (n=9) treated with imetelstat Figure 2. Clinical response in Pts 003 and 004 based on changes in paraprotein level. Dashed line represents off study evaluation. Figure 2. Clinical response in Pts 003 and 004 based on changes in paraprotein level. Dashed line represents off study evaluation. Disclosures: Huff: Celgene: Scientific Advisory Board Other; Novartis: Consultancy. Jones:Cytomedix: Royalties, Royalties Patents & Royalties. Reddy:Geron Corp.: Employment. Nishimoto:Geron Corp: Employment. Stuart:Geron Corp: Consultancy, Employment; OncoMed Pharmaceuticals: Consultancy. Kelsey:Geron Corp: Employment. Matsui:Geron Corp.: Research Funding.

The function of tubulin acetylation in the context of cancer initiation and progression remains unknown. In studying the frequency of primary cilia in early pancreatic cancer, we discovered an increase in acetylated alpha-tubulin labeling (AcTub) labeling in murine PanIN, even in the absence of primary cilia. We therefore hypothesized that human pancreatic ductal adenocarcinoma (PDAC) cells may upregulate expression of AcTub as they progress from noninvasive to invasive and metastatic disease. Performing confocal microscopy on a human PanIN (n=23) and metastatic PDAC (n=10) tissue arrays, we have determined that in 15% of PanIN lesions, 13-30% of the cells have detectable levels of AcTub, with a primarily cytoplasmic/cell membrane pattern of labeling. In contrast, over 80% of metastatic PDAC cells are AcTub-positive. Surprisingly, high-resolution 3-D confocal imaging of both fixed and unfixed human PDAC cell lines demonstrated immunoreactive AcTub to be present on the cell surface of unfixed cells, as previously reported in human leukemia cells (Quillen, JCB 1985). Furthermore, TEM imaging of cancer cells revealed the expression of AcTub on the cell membrane. To evaluate the functional significance of this finding, we developed a FACS-based strategy to isolate viable PDAC cells based on levels of cell surface AcTub. Applying this strategy to four different human pancreatic cancer cell lines and three early passage human xenografts, we have determined that 5-10% of human pancreatic cancer cells have detectable levels of cell-surface AcTub. PDAC cells positive for cell surface AcTub are markedly enriched in cells also positive for either CD133 or CD24/CD44, representing putative pancreatic cancer stem cell populations. We have functionally analyzed the tumor-initiating capacity of cell surface AcTubHI cells using in vitro tumor sphere experiments. In this assay, cell surface AcTubHI cells show a statistically significant 100-fold increase in tumor sphere-forming capacity relative to cell surface AcTublow cells. Tumor sphere-forming capacity was significantly reduced by neutralizing anti-AcTub antibodies, as well as by an siRNA targeting α-tubulin K40 acetyltransferase (αTAT). We further evaluated the functional significance of cell surface AcTub by analyzing the in vivo tumor initiating capacities of cell surface AcTubHI and AcTublow cells from a human PDAC xenograft. Cell surface AcTubHI cells exhibited significantly increased tumor initiating frequency relative to the AcTublow population (**p

Background: The Hedgehog (Hh) signaling pathway plays a pathogenic role in many human cancers, including pancreatic adenocarcinoma, and several pathway antagonists have begun clinical testing. All of these novel agents target Smoothened (Smo), a key regulator of Hh signaling, but reported emergence of resistance mutations questions the durability of this approach. Oxysterols, oxidized derivatives of cholesterol, can modulate Hh signaling either through direct interaction with Smo or via cross-talk with liver X receptors (LXR) that serve as their natural receptors. Thus, oxysterols may represent mechanistically novel Hh inhibitors, especially if they involve LXRs. Methods: We examined the effects of LXR activation on the in vivo growth of pancreatic cancer. Nude mice bearing pancreatic cancer xenografts derived from primary clinical specimens were treated with a vehicle control, gemcitabine, the non-steroidal LXR agonist TO901317, or the combination of gemcitabine and TO901317 for 4-6 weeks. The LXR agonist TO901317 was used since it lacks the ability to directly bind to Smo. Tumor volumes were measured to assess response, and LXR and Hh pathway activities were quantified by real-time RT-PCR for Hh (GLI1, PTCH1) and LXR (ABCA1) target gene expression. Human specific primers were used to detect changes in tumor cells, whereas mouse-specific primers were used to study stroma. Intratumoral levels of gemcitabine triphosphate were quantified by LC-MS-MS. Results: Administration of TO901317 alone did not impact tumor growth compared to control treated animals similar to previous findings with Smo antagonists. Treatment with gemcitabine alone decreased tumor growth, However the addition of TO901317 to gemcitabine significantly enhanced this effect to promote tumor regression. In vivo treatment with TO901317 successfully activated LXR in both human tumor cells and murine stromal cells as evidenced by increased expression of ABCA1. Moreover, TO901317 inhibited expression of the Hh targets GLI1 and PTCH1 5- and 2-fold, respectively, in stromal cells and 5-fold in tumor cells. Previous studies (Olive et al, Science, 2009) demonstrated that Hh pathway inhibition improves drug delivery in vivo. Therefore, we quantified intratumoral levels of gemcitabine triphosphate, the active metabolite of gemcitabine, and found that it was significantly elevated in tumors exposed to TO901317 (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3494. doi:1538-7445.AM2012-3494

Reversal of gene promoter DNA hypermethylation and associated abnormal gene silencing is an attractive approach to cancer therapy. The DNA methylation inhibitors, decitabine (5-aza-2′-deoxycytidine) and azacitidine (5-azacytidine) are proving efficacious clinically for hematological neoplasms, especially at lower, less toxic, doses. Experimentally, high doses induce rapid DNA damage and cytotoxicity, but these may not explain the prolonged time to response often seen in patients. We now show that transient exposure of cultured and primary leukemic and epithelial tumor cells to decitabine or azacitidine at clinically-relevant nanomolar doses, without causing immediate cytotoxicity, produces a “memory” for anti-tumor responses, including potent inhibition of subpopulations of cancer, stem-like cells which often resist other therapies. These inhibitory effects are accompanied by sustained decreases in genome-wide promoter DNA methylation with associated gene re-expression, and anti-tumor changes in multiple key cellular regulatory pathways, such as cell cycle events mediated through FOXM1, cell invasion and motility, and granulocyte and breast cancer cell maturation. Notably, most of the key pathways altered by decitabine or azacitidine involve high priority targets for pharmacologic anti-cancer strategies, which provides molecular basis for possible combination therapies. Thus, low dose decitabine and azacitidine regimens may potentially have broad applicability for cancer management. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 995. doi:1538-7445.AM2012-995

Specific expression of Bruton's tyrosine kinase (Btk) in osteoclasts (OC), but not osteoblasts (OB), suggests its role in osteoclastogenesis. Since Btk has not been characterized in multiple myeloma (MM) and Waldenström Macroglobulinemia (WM), we investigate effects of PCI-32765, an oral, potent, and selective Btk inhibitor with promising clinical activity in B-cell malignancies, on OC differentiation and function within MM bone marrow (BM) milieu, as well as on MM and WM cancer cells. In CD14+ OC precursor cells, PCI-32765 abrogated RANKL/M-CSF-induced Btk activation and downstream PLCγ2, resulting in decreased number of multinucleated OC (>3 nuclei) by tartrate-resistant acid phosphatase (TRAP) staining and inhibition of TRAP5b (ED50 = 17 nM), a specific mature OC marker. It induced defective bone resorption activity, as evidenced by diminished pit formation on dentine slices. Lack of effect of Dexamethasone on osteoclastic activity was overcome by combination of Dexamethasone with PCI-32765. PCI-32765 potently downregulates cytokine and chemokine secretion from OC cultures, i.e., MIP1α, MIP1α, IL-8, TGFβ1, RANTES, APRIL, SDF-1, and activin A (ED50 = 0.1-0.48 nM). It significantly decreased IL-6, SDF-1, MIP1α, MIP1α, and M-CSF in 2-week cultures of CD138-negative cells from active MM patients, associated with decreased TRAP staining. In MM and WM cells, immunoblotting analysis showed Btk expression in higher percentage of CD138+ myeloma cells from patients (4 out of 5 samples) than MM cell lines (5 out of 9 cell lines), whereas microarray analysis demonstrated increased expression of Btk and its downstream signaling components in WM cells than in CD19+ normal BM cells (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2934. doi:1538-7445.AM2012-2934

Despite its rarity, multiple myeloma is an ideal testing ground for cancer biology, says William Matsui.

Abstract 997 Adoptive T cell therapy requires the infusion of highly tumor specific T cells capable of trafficking to the tumor site, killing the tumor and persisting over time. MILs (as compared to peripheral blood T cells) possess many of these features (Noonan, Ca, Res. 2006). We here report the initial results from our Phase I/II trial. 22 patients were transplanted with a Melphalan-200. 19 with a CD34-selected product, 1 unselected autologous stem cells and 2 auto-BMT. 12 underwent SCT as initial consolidation and 10 patients with relapsed disease that were heavily pre-treated with an average of 3.2 prior therapies. Mean ISS score was 2.0. Average age was 56 (29–71). MILs were expanded with anti-CD3/CD28 beads for 7 days (avg. fold expansion 48.5 and avg. cell dose 2.8×10e7 CD3/kg). MILs were infused on day +3. Patients in a CR were ineligible for this study. The best clinical response included 6CRs (27%), 10PRs (45%). Autologous GVHD which was observed in 32% of patients, was limited to the skin and required no treatment. Development of this syndome did not correlate with improved clinical outcomes. Lymphoid recovery was rapid with a mean absolute lymphocyte count (ALC) on day 15 of 886 cells/ul. The laboratory immune monitoring studies were only performed in the bone marrow – the disease site and not in the peripheral blood. The percent CD3+ T cells in the BM was 13% at baseline with a CD4:CD8 ratio of 1:3. By day 60, BM T cell reconstitution was complete and showed an inversion of the CD4:8 ratio of 0.39 that persisted through day 360. At baseline, there were more CD4 effector memory (CD4EM) (CD62L−/CD45RO+) than CD4 central memory (CD4CM) (CD62L+/CD45RO+) (43% vs 27%). The CD4CM population peaked on day 60 at 43% and persisted through 1 year, CD4EM remained unchanged, and the CD4Effector decreased from 19.8% to 5.6%. The CD8 subpopulations remained unchanged from pre-SCT to 1 year post-SCT. Treg numbers doubled from harvest to post-SCT consistent with previous studies showing a greater number of Tregs in healthy BM compared to MM-BM. Activated T cells (CD69+) doubled from pre- to post-SCT. IFNγ production in both CD4 and CD8 cells more than doubled compared to pre-SCT and was maintained at 1 year suggesting the persistence of the infused MILs. The immune monitoring in the BM based on clinical responses revealed that patients achieving a CR/PR showed greater CD8 numbers at day +60 compared to stable disease (SD) or progressive disease (PD)(14.5% vs. 7.6%, respectively) and inversion of the CD4/CD8 ratio at 1 year (0.55 vs 1.26). In patients with SD or PD, the immune infiltrate in the BM was characterized by a large numbers of effector and effector-memory T cells and few CD8CM at baseline. CR patients possessed the fewest CD8Effector and the most CD8CM with persistence of CD8CM out to one year. These patients also showed a greater expansion in IFNγ cells as well as a greater amount of IFNγ production at each time point. Importantly, tumor-specific IFNγ production of CD3 cells in the BM was predictive of a clinical response. Patients achieving a CR showed twice the antigen-specific IFNγ production compared to all other groups at day 180 which persisted through 1 year. Analyses of additional immune subsets will be discussed in greater detail. This is the first reported trial utilizing activated MILs as a source of tumor specific T cells. We have demonstrated the ability to effectively expand MILs ex vivo and provide an analysis of the parameters of immune reconstitution demonstrating that the clinical outcome correlated with the robustness of the immune response in the BM. Disclosures: Luznik: Otsuka Pharmaceuticals: Research Funding.

Abstract 2954 Multiple myeloma (MM) cancer stem cells (CSCs) possess both enhanced tumorigenic potential and relative drug resistance suggesting they play a major role in disease relapse and progression. Therefore, a better understanding of the processes regulating MM CSCs may lead to the development of novel therapies that prevent tumor regrowth and improve long-term outcomes. Normal stem cells are tightly regulated by factors within the local microenvironment that include both soluble factors and direct contact with accessory cells. However, external factors regulating MM CSCs have not been identified. Recent studies have demonstrated that stromal cells in the MM bone marrow microenvironment secrete growth differentiation factor 15 (GDF15), a member of the TGF-b family. We initially studied the role of this cytokine in the pathogenesis of MM by examining circulating GDF15 levels in MM patients. Compared to healthy volunteers, we found that median GDF15 levels were significantly increased in MM patients (821 vs. 390 pg/ml; n=16; p Disclosures: No relevant conflicts of interest to declare.

Abstract 883 Specific expression of Bruton's tyrosine kinase (Btk) in osteoclasts (OC), but not osteoblasts (OB), suggests its role in regulating osteoclastogenesis. Although Btk is critical in B cell maturation and myeloid function, it has not been characterized in plasma cell malignancies including multiple myeloma (MM) and Waldenström Macroglobulinemia (WM). We here investigate effects of PCI-32765, an oral, potent, and selective Btk inhibitor with promising clinical activity in B-cell malignancies, on OC differentiation and function within MM bone marrow (BM) microenvironment, as well as on MM and WM cancer cells. We further define molecular targets of Btk signaling cascade in OCs and MM in the BM milieu. In CD14+ OC precursor cells, RANKL and M-CSF stimulate phosphorylation of Btk in a time-dependent fashion; conversely, PCI-32765 abrogates RANKL/M-CSF-induced activation of Btk and downstream PLCγ2. Importantly, PCI-32765 decreased number of multinucleated OC (>3 nuclei) by tartrate-resistant acid phosphatase (TRAP) staining and the secretion of TRAP5b (ED50 = 17 nM), a specific mature OC marker. It increased size of OCs and number of nuclei per OC, with significantly defective bone resorption activity as evidenced by diminished pit formation on dentine slices. Moreover, lack of effect of Dexamethasone on OC activity was overcome by combination of Dexamethasone with PCI-32765. PCI-32765 significantly reduced cytokine and chemokine secretion from OC cultures, including MIP1α, MIP1β, IL-8, TGFβ1, RANTES, APRIL, SDF-1, and activin A (ED50 = 0.1–0.48 nM). It potently decreased IL-6, SDF-1, MIP1α, MIP1β, and M-CSF in CD138-negative cell cultures from active MM patients, associated with decreased TRAP staining in a dose-dependent manner. In MM and WM cells, immunoblotting analysis confirmed a higher Btk expression in CD138+ cells from majority of MM patients (4 out of 5 samples) than MM cell lines (5 out of 9 cell lines), whereas microarray analysis demonstrated a higher expression of Btk and its downstream signaling components in WM cells than in CD19+ normal bone marrow cells. PCI-32765 significantly inhibits SDF-1-induced adhesion and migration of MM cells. It further blocked cytokine expression (MIP1a, MIP-1β) at mRNA level in MM and WM tumor cells, correlated with inhibition of Btk-mediated pPLCγ2, pERK and NF-kB activation. Importantly, PCI-32765 inhibited growth and survival triggered by IL-6 and coculture with BM stromal cells (BMSCs) or OCs in IL-6-dependent INA6 and ANBL6 MM cells. Furthermore, myeloma stem-like cells express Btk and PCI-32765 (10–100 nM) blocks their abilities to form colonies from MM patients (n=5). In contrast, PCI-32765 has no adverse effects on Btk-negative BMSCs and OBs, as well as Btk-expressing dendritic cells. Finally, oral administration of PCI-32765 (12 mg/kg) in mice significantly suppresses MM cell growth (p< 0.03) and MM cell-induced osteolysis on implanted human bone chips in a humanized myeloma (SCID-hu) model. Together, these results provide compelling evidence to target Btk in the BM microenvironment against MM and WM., strongly supporting clinical trials of PCI-32765 to improve patient outcome in MM and WM. Disclosures: Chang: Pharmacyclics Inc: Employment. Buggy:Pharmacyclics, Inc.: Employment, Equity Ownership. Elias:Pharmacyclics Inc: Consultancy. Treon:Millennium: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Genentech: Honoraria. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: 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; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Actelion: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

The highly conserved Hedgehog (Hh) signaling pathway is normally required for both embryonic development and adult tissue homeostasis and post-injury repair. In vertebrates, signaling is activated by the binding of one of the three ligands, Sonic (SHh), Desert (DHh), and Indian (IHh) Hedgehog, to Patched (Ptch), a 12-pass transmembrane spanning receptor. In the absence of ligand, Ptch represses Smoothened (Smo), a 7-pass transmembrane protein and positive regulator of pathway activity. Following ligand binding, Smo is de-pressed allowing the expression and post-translational processing of Gli transcription factors. Three forms of Gli exist. Gli1 induces and Gli3 represses target gene expression, whereas Gli2 can act either positively or negatively on transcription depending on post-transcriptional and post-translational modifications. Ultimately, the biological activity of the pathway is dictated by the balance between activating and repressive forms of the Glis at target gene promoters (1). The identification of somatic PTCH1 mutations in patients with Gorlin syndrome and basal cell nevus syndrome who carry an increased risk of developing advanced basal cell carcinoma (BCC), medulloblastoma, and rhabdomyosarcoma initially implicated Hh pathway dysregulation in human carcinogenesis. Further support was provided by the detection of frequent Ptch1 and Smo mutations in sporadic BCCs and medulloblastoma as well as the ability of conditional Ptch loss-of-function or Smo and Gli gain-of-function mutations to recapitulate these diseases in transgenic mice (2). In addition to BCC and medulloblastoma, aberrant Hh signaling has been found in many other cancers, but pathway activity results from increased levels of Hh ligand rather than mutations within pathway components. More recently, multiple types of pathway activation have been described within these ligand-dependent tumors (3). Autocrine and/or juxacrine signaling in which tumor cells both produce and respond to Hh ligand represents the simplest model and has been described in small cell lung, pancreatic, colon, and metastatic prostate carcinomas as well as glioblastoma and melanoma. More recently, Hh signaling has been found to act in a paracrine fashion in which cells that produce Hh ligands are distinct from those responding with pathway activation. Paracrine signaling has been demonstrated in B cell lymphomas and multiple myeloma (MM) in which bone marrow or lymph node stromal cells produce Hh ligand that activates signaling in tumor cells (4). Increased Hh signaling in these settings has been found to promote a myriad of effects including enhanced tumor cell proliferation, survival, and chemoresistance. In pancreatic and colon cancers, an alternative mode of paracrine Hh signaling exists in which tumor cells secrete Hh ligands that induce pathway activity within non-malignant stromal cells. Here, Hh signaling within the tumor microenvironment leads to the secretion of growth factors and cytokines that in turn increase tumor cell survival or angiogenesis (5). The Hh pathway has also been found to regulate cancer stem cells (CSCs) in a number of human diseases including glioblastoma, breast cancer, colon cancer, pancreatic adenocarcinoma, MM and chronic myeloid leukemia (CML). Self-renewal is a defining property of CSCs required for maintenance of the malignant clone, and studies involving both human tumors and mouse models of cancer have found that this process is dependent on active Hh signaling. In MM, we found that Hh signaling results in CSC expansion whereas pathway inhibition induces terminal differentiation and loss of clonogenic growth potential (6). Therefore, Hh pathway activity appears to regulate CSC fate decisions that include self-renewal and differentiation. Increasing data have also implicated a role for CSCs in the development of metastatic disease, and in pancreatic and colorectal carcinomas, active Hh signaling induces the epithelial to mesenchymal transition within CSCs to enhance their invasive and migratory properties (7). The emerging role of Hh signaling in human cancers has fueled efforts to develop Hh pathway inhibitors. The discovery that cyclopamine, a steroidal alkyloid derived from Veratrum californicum, binds to Smo and inhibits Hh signaling provided early evidence that the pathway could be pharmacologically targeted (8). Large-scale compound screens have successfully identified several other Smo antagonists, and despite their unbiased nature, most agents bind to and inhibit Smo even though they are structurally distinct from one another and cyclopamine. These agents have moved into clinical testing and the first trial targeting the Hh pathway used one such Smo antagonist, vismodegib (GDC-0449; Genentech/Roche) in patients with advanced solid tumors. Efficacy in patients with advanced BCC was detected early in this trial, presumably because of the high frequency activating mutations, and it was subsequently expanded to specifically study BCC. Out of a total of 33 patients with locally advanced or metastatic BCC 55% demonstrated clinical responses that included 2 complete remissions (9). Reported toxicities were mild, and the most common events were mild loss of taste, hair loss, weight loss and hyponatremia. A transient subjective tumor response was also reported in a single medulloblastoma patient treated with vismodegib. Similar findings in advanced BCC and medulloblastoma have been reported with other Smo antagonists including IPI-926 (Infinity), LDE225 (Norvartis) and BMS-833923 (Bristol-Meyers Squibb/Exelixis). Initial results with Smo antagonists have been encouraging, but alternative means of inhibiting Hh signaling may be useful for multiple reasons. These include the development of resistance mutations in Smo, the identification of mutations in Hh pathway components that act downstream of Smo (e.g., mutation of the negative pathway regulator Sufu or amplifications in Glis), and direct induction of Gli1 activity by transforming events (e.g., Ews-Fli in Ewing sarcoma or mutant KRas in pancreatic cancer). To this end, novel compounds targeting signal transduction components beyond Smo have been identified. Since mutations in Hh pathway components have not been described in most cancers, the inhibition of ligand binding may repress Hh signaling and the ligand neutralizing monoclonal antibody 5E1 has demonstrated anti-tumor activity in several disease models. In addition, the novel agent robotnikinin has been found to prevent Hh ligands from activating Ptch (10). Since pathway activation cumulates in transcriptional activation by Glis, small molecules modulating Gli activity (GANT-58 and GANT-61) may be useful (11). Furthermore, compounds inhibiting pathway activity by altering the post-translational processing and cellular localization of Gli1 and Gli2 (HPI-1, HPI-2, HPI-3, arsenic) or inhibiting the formation of primary cilia required for Hh signal transduction (HPI-4) have been identified (12, 13). Finally, it is possible that the interaction of other cellular pathways, such as PI3K/AKT, MAPK, TGF-β, and Liver × receptors may also target aberrant Hh signaling. A role for aberrant Hh signaling pathway is emerging in a wide variety of human malignancies that include multiple roles in disease initiation, relapse, and progression. Therefore, improved understanding of the basic mechanisms that govern Hh signal transduction, the interaction of Hh signaling with other cellular pathways, and the precise role of Hh in specific malignancies should improve the anti-tumor activity of these novel approaches. References: 1. Ruiz i Altaba A, Mas C, Stecca B. The Gli code: an information nexus regulating cell fate, stemness and cancer. Trends Cell Biol. 2007;17:438–47. 2. Pasca di Magliano M, Hebrok M. Hedgehog signalling in cancer formation and maintenance. Nat Rev Cancer. 2003;3:903–11. 3. Merchant AA, Matsui W. Targeting Hedgehog — a cancer stem cell pathway. Clin Can Res. 2010;16:3130–40. 4. Dierks C, Grbic J, Zirlik K, Beigi R, Englund NP, Guo G-R, et al. Essential role of stromally induced hedgehog signaling in B-cell malignancies. Nat Med. 2007;13:944–51. 5. Theunissen J-W, de Sauvage FJ. Paracrine Hedgehog signaling in cancer. Cancer Res. 2009;69:6007–10. 6. Peacock CD, Wang Q, Gesell GS, Corcoran-Schwartz IM, Jones E, Kim J, et al. Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma. Proc Natl Acad Sci USA. 2007;104:4048–53. 7. Varnat F, Duquet A, Malerba M, Zbinden M, Mas C, Gervaz P, et al. Human colon cancer epithelial cells harbour active HEDGEHOG-GLI signalling that is essential for tumor growth, recurrence, metastasis and stem cell survival and expansion. Embo Mol Med. 2009;1. 8. Taipale J, Chen JK, Cooper MK, Wang B, Mann RK, Milenkovic L, et al. Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine. Nature. 2000;406:1005–9. 9. Von Hoff D, Lorusso P, Rudin C, Reddy J, Yauch R, Tibes R, et al. Inhibition of the Hedgehog pathway in advanced basal-cell carcinoma. N Engl J Med. 2009. 10. Stanton BZ, Peng LF, Maloof N, Nakai K, Wang X, Duffner JL, et al. A small molecule that binds Hedgehog and blocks its signaling in human cells. Nat Chem Biol. 2009;5:154–6. 11. Lauth M, Bergström A, Shimokawa T, Toftgård R. Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists. Proc Natl Acad Sci USA. 2007;104:8455–60. 12. Hyman JM, Firestone AJ, Heine VM, Zhao Y, Ocasio CA, Han K, et al. Small-molecule inhibitors reveal multiple strategies for Hedgehog pathway blockade. Proc Natl Acad Sci U S A. 2009;106:14132–7. 13. Kim J, Lee J, Gardner D, Beachy P. Arsenic antagonizes the Hedgehog pathway by preventing ciliary accumulation and reducing stability of the Gli2 transcriptional effector. Proc Natl Acad Sci U S A. 2010;107:13432. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr CN06-04.

Pancreatic adenocarcinoma is a highly aggressive disease with a propensity for metastasis early in its course. Pancreatic cancer stem cells (CSCs) have been thought to play a key role in this process. We previously found that pancreatic CSCs express increased levels of aldehyde dehydrogenase (ALDH) and at the time of diagnosis the presence of ALDH+ cells in primary tumors is associated with worse overall survival. The development of CSC-targeting strategies have largely focused on intrinsic signaling pathways, but normal stem cells are primarily regulated by the extracellular stem cell niche that consists of non-malignant cells and specific extracellular matrix (ECM) proteins. The densely fibrotic desmoplastic reaction (DR) is a common feature of pancreatic tumors, thus we examined the role of the ECM on pancreatic CSCs. We initially studied the distribution of ALDH+ cells in primary pancreatic tumors by immunohistochemistry and found that they were more frequently located at the invasive edge of tumors adjacent to the DR compared to the rest of the tumor ( Our data suggest that the interaction between pancreatic CSCs and the DR results in enhanced tumorigenicity and metastatic potential. Furthermore, the disruption of FAK signaling can inhibit pancreatic CSC function and may serve as a novel therapeutic strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2446. doi:10.1158/1538-7445.AM2011-2446

Primary cilia are microtubule rich projections eminating from the apical surface of epithelial cells. Primary cilia are responsible for the transduction of growth factor and morphogenetic signaling pathways, including the hedgehog, wnt and platelet-derived growth factor pathways. Initial observations in the field of pancreatic cancer have suggested the absence of primary cilia in human pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma. While absent in the majority of pancreatic cancer cells, primary cilia have been identified in differentiated acinar cells undergoing an acinar to ductal metaplasia. This metaplasia is well characterized in pancreatic injury and regeneration models and accelerates tumor formation in the presence of acinar specific KrasG12D mutations. Thus, we are addressing the hypothesis that primary cilia, while not expressed in bulk tumor populations, are maintained in pancreatic tumor-initiating cells. To address the hypothesis, we have used confocal microscopy to quantify the percentage of cells within low grade human PanIN lesions that express a primary cilium. Performing confocal microscopy on a human PanIN tissue array (n=23), we have determined that in 15% of PanIN lesions, 13-30% of the cells are expressing a primary cilium, as is evidenced by staining for acetylated alpha tubulin. We have also developed a sorting strategy using an acetylated alpha tubulin antibody to isolate ciliated cells by Fluorescence Activated Cell Sorting (FACS) analysis. Using this protocol, we have discovered that human pancreatic cancer cells bearing a primary cilium are markedly enriched in cells also positive for either CD133 or CD24/CD44, representing putative pancreatic cancer stem cell populations. We have functionally analyzed the tumor-initiating capacity of ciliated vs non-ciliated cells using tumor sphere experiments. In this assay, ciliated cells show a significant increase (100 fold) in tumor sphere-forming capacity, suggesting that the presence of a primary cilium may mark a pancreatic cancer tumor initiating population. These data are informative regarding the earliest initiating events in pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2449. doi:10.1158/1538-7445.AM2011-2449

It has been firmly established that cancer gene expression partly mimics the gene expression signature in embryonic stem cells (ESC). Since most cancers arise in adult stem or progenitor cells, rather than ESC, we now compare cancer chromatin to both embryonic and adult cell renewal systems to understand the relationships between DNA hypermethylation, gene expression, and chromatin states. DNA hypermethylation at CpG island promoters of hundreds of genes, including classic tumor suppressors, is a major modulator of gene expression in human cancers. Past studies suggest ∼ 50% of these are frequently marked by polycomb complex (PcG) transcriptional repressors, but not DNA methylation, in embryonic stem cells (ESC). In ESC, PcG occupancy is predominantly in the context of “bivalent chromatin”, wherein the active transcription mark H3K4me3 and the repressive PcG mark H3K27me3 are simultaneously present. Genes so marked are in a low, but poised, transcription state important for stemness and self-renewal, characteristics shared with tumor cells. Using whole genome ChIP-seq and DNA methylation arrays, we find between 70 to 80% of genes with DNA hypermethylation in cancer have bivalent chromatin not only in ESC, but also in adult stem cells. For these genes, there appears to be an epigenetic switch in cancer wherein bivalent chromatin is replaced by DNA hypermethylation resulting in tighter repression of gene expression. Many of these DNA methylated cancer genes are constituents of the recently proposed “PRC module” of the “ESC cancer signature.” Our data suggest a “DNA methylation module” that recapitulates the “stem cell signature” and that DNA hypermethylation may be a key mechanism that confers cell-renewal and stemness to tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-174. doi:10.1158/1538-7445.AM2011-LB-174