ALBERT

Description: Key Points The tumor microenvironment drives myeloma cell clonogenic growth and self-renewal through GDF15.

Description: Aberrant self-renewal is a hallmark of cancer and is central to the initiation, maintenance and relapse of clinical disease. The cellular processes responsible for self-renewal have not been delineated in most human cancers, but it is likely that conserved pathways required for the regulation of normal stem cells are involved. Notably several highly conserved signaling pathways that regulate stem cell fate decisions during embryonic development, such as Notch, Hedgehog (Hh) and Wingless (Wnt), are inappropriately activated in a wide variety of human cancers. We recently demonstrated that the Hh signaling pathway is required for the maintenance of cancer stem cells in the plasma cell malignancy, multiple myeloma. Since myeloma stem cells phenotypically resemble normal B cells, we hypothesized that aberrant Hh signaling is a feature of other B cell malignancies. We studied established human cell lines derived from patients with classical Hodgkin lymphoma (L428, KM-H2), diffuse large B cell NHL (HT, Pfeiffer, RL, and Hs 602), and mantle cell NHL (Granta 519, Jeko-1, Rec-1) and found that expression of the Hh signaling pathway components PATCHED (PTCH), SMOOTHENED (SMO), and GLI1, 2 or 3 by RT-PCR was markedly elevated compared to normal B cells in the majority of cell lines from each subtype of lymphoma. In order to examine the functional role of Hh signaling on human lymphomas, cells were treated with recombinant human sonic Hh ligand (SHh) or the naturally occurring inhibitor of SMO, cyclopamine, followed by evaluation of clonogenic growth in methylcellulose. Resulting colony formation was significantly increased in response to activating SHh ligand, whereas treatment with cyclopamine significantly inhibited clonogenic recovery. Similarly, the inhibition of pathway signaling by neutralizing anti-Hh antibodies limited colony formation suggesting that ligand binding by PTCH was required for pathway activation similar to other non-Gorlins tumors such as small cell lung cancer, pancreatic carcinoma, and metastatic prostate cancer that lack mutation of pathway components. Furthermore, we examined the activity of a novel semi-synthetic cyclopamine analogue, IPI-609, and found that it also limited clonogenic lymphoma growth. The effects of IPI-609 were highly specific as the clonogenic recovery of cell lines lacking expression of Hh pathway components was not affected by treatment. Our previous studies in multiple myeloma have suggested that cancer stem cells can be identified by their relatively higher activity of the intracellular enzyme retinaldehyde dehydrogenase (ALDH) similar to normal hematopoietic and neural stem cells. We found that high ALDH activity could also identify rare cell populations with greater clonogenic growth potential compared to ALDHlow/neg cells in the majority of lines and treatment with cyclopamine or IPI-609 significantly reduced the relative proportion of ALDHhigh cells. Therefore, the Hh signaling pathway may represent a novel therapeutic target in human lymphomas. Moreover, novel Hh inhibitors, such as IPI-609, may inhibit highly clonogenic lymphoma cancer stem cells responsible for disease relapse.

Description: Multiple myeloma (MM) is an incurable hematological malignancy characterized by recurrent chromosomal translocations. The t(4;14)(p16;q32) is associated with the worst prognosis of any patient subgroup in MM, although the basis for this poor prognosis is unknown. The t(4;14) is unusual in that it involves two potential target genes on chromosome 4: fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain (MMSET). MMSET is universally over-expressed in t(4;14) MM, whereas FGFR3 expression is lost in one third of cases, suggesting a role for MMSET in myeloma pathogenesis. 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. Using homologous recombination-mediated gene targeting, we disrupted the N-terminal and full-length isoforms of MMSET in t(4;14)+ KMS-11 MM cells. Disruption of the translocated MMSET allele revealed that this allele accounts for most of the MMSET transcription in t(4;14) MM cells. Accordingly, selective targeting of the translocated allele, but not the non-translocated allele, led to reduced colony formation in methylcellulose and reduced tumor formation in nude mouse xenografts. Down-regulation of MMSET expression in t(4;14) MM cell lines by stable RNA interference (RNAi) led to a slower growth in liquid culture, a significant reduction in colony formation in methylcellulose, and decreased tumorigenicity in vivo. Additionally, MMSET knockdown led to partial cell cycle arrest of adherent MM cells and reduced the ability of MM cells to adhere to extracellular matrix. Cells with targeted disruption or knockdown of MMSET exhibited changes in expression levels of potential target genes, including several adhesion molecules. These results provide the first direct evidence that translocation-mediated overexpression of MMSET plays a critical role in t(4;14) MM and suggest that therapies targeting this gene could impact this particular subset of poor-prognosis patients.

Description: We recently demonstrated that multiple myeloma (MM) is organized in a hierarchical manner in which clonogenic MM progenitors or stem cells resembling post-germinal B cells give rise to MM plasma cells (PC). To study the potential biologic differences between MM stem cells and MM PC, we examined each cellular subset for characteristics found in normal stem cells as well as their responses to various antitumor agents. The human MM cell lines RPMI 8226 and NCI-H929 were initially studied as we previously found that they recapitulate clinical MM specimens and consist of distinct cell populations based on the expression of the PC surface antigen CD138; CD138+ cells resemble typical MM PC, whereas CD138neg cells express B cell surface antigens and have greater clonogenic capacity. Examination of these cellular subpopulations by flow cytometry demonstrated that CD138neg cells were smaller and less granular by light scatter than CD138+ PC and expressed higher levels of the intracellular enzyme aldehyde dehydrogenase that is present in normal hematopoietic progenitors with self-renewal potential. Furthermore, cells expressing the side population phenotype after staining with the DNA binding dye Hoechst 33342 were exclusively CD138neg. We also investigated the effects of different clinically applicable agents on CD138+ and CD138neg cells. CD138+ and CD138neg cells isolated from RPMI 8226 and NCI-H929 cells by fluorescence activated cell sorting were treated with dexamethasone (dex, 100nM), bortezomib (velcade, 10nM), CC5013 (revlimid, 1μM), rituximab (10μg/ml) or alemtuzumab (campath,10μg/ml) for 72 hours followed by plating in methylcellulose to assess clonogenic capacity. CD138+ PC were significantly inhibited by dex (27 ± 11% recovery compared to untreated control cells), velcade (14 ± 6%) and revlimid (44 ± 27%), whereas rituximab (92 ± 25%) and campath (97 ± 18%) had little activity. In contrast, clonogenic growth of CD138neg cells was not significantly inhibited by dex (82 ± 19%), velcade (88 ± 29%), or revlimid (91 ± 14%), but was significantly decreased by rituximab (63 ± 22%) and campath (47 ± 27%). Similarly, clonogenic MM growth of CD138neg cells from 4 clinical MM samples was not affected by dex (84 ± 9%), velcade (82 ± 24%), or revlimid (93 ± 11%), but was significantly inhibited by rituximab (19 ± 7%) or campath (15 ± 11%). Clonogenic MM precursors may be distinguished from MM PC by a variety of biological parameters typically expressed by normal stem cells. Furthermore, these cellular subsets have different susceptibilities to a variety of clinical agents, and agents with activity against MM PC may be ineffective against MM stem cells. Moreover, agents without activity agasint MM PC may have major activity against MM stem cells. The divergent sensitivities of MM stem cells and PC may explain the dramatic, but transient, responses seen with many agents. Therapeutic strategies that result in long-term remissions may require the inhibition of both MM PC to reduce clinical symptoms and MM stem cells responsible for relapse.

Description: 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.

Description: Telomerase activity (TA) is required within normal cells capable of long-term replication, including stem cells and is upregulated in many cancers. In the absence of TA or presence of TA inhibitors, the progressive shortening of telomeres ultimately results in cellular senescence and/or apoptosis. These observations support that TA inhibitors represent a novel class of anti-tumor agents. Much evidence suggests that human cancers display a hierarchical cellular organization that mirrors normal tissues. Cancer stem cells (CSC) are derived from the malignant transformation of normal stem cells and progenitors and retain the capacity to self-renew. Moreover, CSC give rise to differentiated tumor cells that form the bulk of the tumor mass, but have little or no capacity for long-term proliferation. We recently demonstrated that the malignant CD138+ plasma cells in multiple myeloma (MM) have limited replicative potential; instead they arise from the differentiation of clonogenic CSC that resemble normal memory B cells (CD138negCD19+CD27+). In addition, several groups have demonstrated that telomerase inhibitors are active against human MM cell lines in vitro and in vivo. We examined TA in CD138+ plasma cells and CD138neg precursors, and studied the effects of telomerase inhibition against both cell populations. We isolated CD138+ and CD138neg cells by FACS from three human MM cell lines (RPMI 8225, NCI-H929, and U266) and measured TA using a PCR-based assay of activity. For each cell line, TA was detectable within both the CD138neg and CD138+ cell populations. GRN163L is a lipid conjugated 13 nucleotide thio-phosphoramidate oligonucleotide that acts as a potent and specific active site inhibitor of telomerase. We found that treatment with GRN163L (0.1–5μM) markedly reduced TA within 48 hours. To examine the effects of telomerase inhibition on clonogenic growth, we continuously cultured CD138+ and CD138neg RPMI 8226 cells with GRN163L (1μM). Cells were collected weekly, washed to remove GRN163L, and then plated in methylcellulose to assess colony formation. We found that GRN163L was active against both CD138+ and CD138neg cells and eliminated the colony forming potential of both by 5 weeks. Similarly, we found that GRN163L inhibited the in vitro clonogenic growth of CD138neg MM CSC isolated from the bone marrow aspirates of patients with MM. These data demonstrate that TA is detectable within both immature MM CSC and mature MM plasma cells, and that CSC from both cell lines and primary clinical samples are targeted by the telomerase inhibitor GRN163L. Therefore, this agent may offer a novel therapeutic approach to myeloma as well as other diseases in which CSC have been identified.

Description: 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

Description: 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.

Description: The identity of the cells responsible for the initiation and maintenance of multiple myeloma (MM) remains unclear largely because of the difficulty growing MM cells in vitro and in vivo. MM cell lines and clinical specimens are characterized by malignant plasma cells that express the cell surface antigen syndecan-1 (CD138); however, CD138 expression is limited to terminally differentiated plasma cells during B-cell development. Moreover, circulating B cells that are clonally related to MM plasma cells have been reported in some patients with MM. We found that human MM cell lines contained small (〈 5%) subpopulations that lacked CD138 expression and had greater clonogenic potential in vitro than corresponding CD138+ plasma cells. CD138- cells from clinical MM samples were similarly clonogenic both in vitro and in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, whereas CD138+ cells were not. Furthermore, CD138- cells from both cell lines and clinical samples phenotypically resembled postgerminal center B cells, and their clonogenic growth was inhibited by the anti-CD20 monoclonal antibody rituximab. These data suggest that MM “stem cells” are CD138- B cells with the ability to replicate and subsequently differentiate into malignant CD138+ plasma cells.

Description: 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.