Absence Of Stroma Research Articles

Integrated Functional Genomic and Pharmacological Characterization of the Kinome of Myeloma Cells

Small molecule kinase Inhibitors represent the main therapeutic modality for targeting aberrant kinase signaling in cancer. The recent emergence of CRISPR/Cas9 based functional genomic tools facilitates the genome-wide assessment of cancer cell dependencies, and evaluation of the relative dependence of tumor cells on diverse types of kinases inhibited by small molecule agents with different specificities, as well as other kinases for which selective inhibitors may not be currently available. In this study, we integrated the phenotypic information from the response of multiple myeloma cell lines to small molecule kinase inhibitors (16 MM cell lines, cultured in the presence vs. absence of stromal cells, using a panel of 273 kinase inhibitors (100nM, 24-72 h exposure; targeting a total of 43 known primary oncogenic targets) vs. CRISPR-based functional data on the relative dependency of MM lines on the respective targets of these inhibitors. The cell-autonomous activity of the various inhibitors was broadly potent for Aurora, PLK, and mTORC1/2 inhibitors, consistent with the high proliferative rates of MM cell lines in vitro and the role of the corresponding genes as broad-spectrum dependencies across not only MM, but also a collection of 300+ cell lines from other neoplasias. We observed virtually no anti-MM in vitro activity for selective inhibitors of c-met, ALK, EGFR, c-kit, PDGFR, VEGFR, Syk, Src, and BTK (even in cell lines with detectable transcripts for the respective kinases):, consistent with these results, the corresponding kinases genes were not essential for in vitro survival and proliferation of MM cell lines in our CRISPR studies. We observed modest to minimal response to selective pharmacological inhibitors for PDK1, PI3K (excluding those that also inhibit mTOR), and Akt. Interestingly, at the functional genomic level, PI3KCA was a prominent dependency (and more pronounced than other PI3K isoforms) for several MM cell lines, while individual isoforms of AKT were typically not essential genes for most or all of the MM cell lines tested, suggesting potential redundancy between these isoforms or feedback loops as bypass mechanisms attenuating the effect of pharmacological inhibitors. Prominent and selective MM kinase dependencies at the functional genomic level include PIM2 (providing indirect explanation for the clinical activity observed in Phase 1 trials in MM of a pan-PIM inhibitor) and IKBKB (consistent with the role of the NFKB pathway in MM). Consistent with prior studies on small inhibitors against IGF1R, its gene is a dependency in most MM lines tested and one of the most recurrent dependencies among receptor tyrosine kinases. Both pharmacological and CRISPR-based results suggest heterogeneous responses of MM cells to loss-of-function of MEK1/2 and, more broadly to diverse members of the MAPK family. Comparison of pharmacological results obtained in the presence vs. absence of stroma, indicate heterogeneous effect of bone marrow stromal cells on MM cell response to inhibitors across different targets and cell lines, including stroma-induced resistance to the same kinase inhibitor for some cell lines vs. sensitization for others. Our results provide novel insights into the relative role of different classes of kinases in MM; the similarities and potential differences in the impact of pharmacological inhibition vs. genomic perturbation of these kinases. These integrated functional genomic and pharmacological studies of the kinome provide a framework for the potential development of inhibitors against previously understudied or underappreciated kinases; and the development of molecular or functional genomic assays that may potentially guide future efforts towards individualized administration of kinase inhibitors in MM. DisclosuresMitsiades:TEVA: Research Funding; Novartis: Research Funding; Janssen/Johnson & Johnson: Research Funding; Ono: Research Funding; Abbvie: Research Funding; Takeda: Other: Employment of family member.

RAR-Alpha Targeting Compounds Overcome Bone Marrow (BM) Stromal Protection of AML By CYP26

All-trans retinoic acid (ATRA) causes terminal differentiation and apoptosis of non-APL AML cells in vitro, but has not proven clinically effective. We have recently shown that BM stroma expressing CYP26 inactivates ATRA and protects leukemia cells from differentiation (Su M et al 2015). ATRA signals through retinoic acid receptors (RARs) α, β and γ and induces not only terminal differentiation of AML, but also upregulation of CYP26, potentially forming an even more protective niche for leukemia. In an effort to decouple these two biological effects and improve differentiation therapy for AML, we investigated the relative contribution of RARα and RARγ to differentiation and CYP26 upregulation.ATRA (pan-RAR agonist), AM80 (strong RARα agonist, weak RARγ), and IRX5183 (RARα-specific agonist; previously known as NRX 195183, Io Therapeutics, Inc.) all induced significant differentiation of NB4 APL cells (i.e., upregulation of myeloid differentiation antigen CD11b and decreased clonogenic activity) at 0.1 μM. In contrast, the RARγ-specific agonist CD437 (0.1 μM) produced no evidence of differentiation. To determine the impact of RARα and RARγ signaling on stromal CYP26B1 levels, we treated murine OP9 BM stroma cells in low serum conditions with the aforementioned agents. ATRA induced a 40.5±30.1 fold upregulation of CYP26B1 at 24h (p=0.02 compared to control) and 24.7±20.4 fold upregulation at 48 hours (p=0.03). CD437 showed progressive upregulation of CYP26B1 (14.4±5.2 fold at 24h and 27.7±8.8 fold at 48h, p<0.01 for both). In contrast, the RARα-specific IRX5183 had only modest effects on CYP26B1 levels (2.9±0.9 fold at 24h and 2.7±0.2 fold at 48h, p<0.01 for both). AM80 showed the highest up-regulation of CYP26B1 levels both at 24h and 48h of treatment (29.1±7.9 and 77±24.4, p<0.01 for both). Since RARγ activation appeared to be dispensable for differentiation, we tested if the two RARα active agents, AM80 and IRX5183, could bypass stromal-mediated protection against ATRA-induced differentiation, as both of these compounds have also been reported to be resistant to CYP26 mediated degradation. Several AML cell lines were cultured in the presence or absence of OP9 BM stroma and treated with ATRA, AM80 or IRX5183 at concentrations of 0.01 to 0.1 μM. We have previously shown that ATRA can induce differentiation of most AML cell lines in stroma-free conditions, but it is inactive in the presence of BM stroma; further, its effect can be restored by inhibition of stromal CYP26 (Su M et al. 2015). Whereas stroma blocked upregulation of CD11b and inhibition of clonogenicity of NB4 cells by ATRA, both AM80 and IRX5183 showed similar activity in the presence or absence of stroma (e.g. AM80 resulted in a fold change CD11b from control of 9.8±5.3 off stroma vs 11.8±8.4 on stroma, p=NS). Stroma also blocked ATRA's activity against NPM1-mutated OCI-AML3 cells (clonogenic recovery from control 6.4±3.3% vs 61.4±12.2% on stroma, p=0.002), but AM80 and IRX5183 were active both in the presence and absence of stroma (e.g. IRX5183 resulted in clonogenic recovery from control of 7.3±2.5% vs 11.2±2.8% on stroma, p=NS). Similar effects were observed using Kasumi-1 core binding factor AML cells.In conclusion, we found that RARα and γ have distinct effects on niche vs AML cells. Stimulation of RARγ does not induce differentiation of AML cells, but leads to the upregulation of stromal CYP26B1, and thus enhanced resistance to ATRA. In contrast, CYP26 resistant, RARα active synthetic retinoids AM80 and IRX5183 are able to differentiate and eliminate AML cells even in the presence of BM stroma regardless of induction of CYP26 expression. We are currently exploring a phase I/II clinical trial using IRX5183 in non-APL AML. DisclosuresChandraratna:Io Therapeutics, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: President.

The Combination of the Novel Syk Inhibitor TAK659 with Decitabine Exerts Synergistic Cytotoxic Effects Against FLT3/ITD Mutated Acute Myeloid Leukemia Cells

Background: Effective treatment regimens for FLT3/ITD+ AML patients are lacking. Recent data on the effects of FLT3 tyrosine kinase inhibition showed promising clinical activity, but outcomes have been stunted by drug resistance. Targeting additional signaling pathways besides FLT3 might therefore offer therapeutic benefit. Spleen tyrosine kinase (syk) is frequently overexpressed and constitutively activated in primary AML FLT3/ITD+ blasts. Pharmacologic inhibition of syk has been associated with antileukemic potential, consisting of inhibition of cell growth and induction of differentiation in AML cells. Hypomethylating agents (DNMTis) such as Decitabine (DEC) have emerged as attractive agents in the treatment of AML, partially due to their proapoptotic and differentiation inducing effects. There is a strong rationale for combining FLT3- and syk- inhibitors with DNMTis for FLT3/ITD+ AML. Here, we investigated whether the antileukemic effects of the novel, dual FLT3/syk inhibitor TAK659 (TAK) can be enhanced by the co-administration of DEC.Methods: Molm14 (M14) cells were incubated in culture medium in the presence and absence of stromal cells (MS5) for 72 hours. TAK or DEC was added in single agent or combined mode at the indicated concentrations. After the incubation period, cell growth was determined by MTT assays. Apoptosis, autophagy and cell cycle status were assessed by FACS analysis. FLT3- and syk- phosphorylation levels were detected by western blotting. Differentiation was assessed by morphology. A combination index (CI) was calculated using Calcusyn Software.Results: TAK exerted dose dependent effects against M14 cells. However, the presence of stromal cells significantly blunted TAK mediated cytotoxicity (50nM: 61.9±1.3% [off stroma] vs. 10.9±3.1% [on stroma], p<.001; 500nM: 91.2±0.5% [off stroma] vs. 17.4±2.5% [on stroma], p<.001, n=3-9; IC50: 40.6nM [off stroma] vs. 3.9µM [on stroma]). Although less marked when stroma was present, TAK combined with DEC yielded synergistically enhanced growth inhibition (off stroma: 50.2±3.9% [TAK659 50nM], 58.1±3.0% [DEC 1µM], 76.7±3.1% [TAK659 50nM+DEC 1µM; CI=0.77]; on stroma: 9.5±3.0%[TAK659 50nM], 42.9±3.1% [DEC1µM], 57.0±5.2%[TAK659 50nM+DEC 1µM; CI=0.62]) compared to single agent treatment (off stroma: p<.001 vs. TAK, p< .001 vs. DEC, on stroma: p<.001 vs.TAK, p=.03 vs. DEC, n=3). In line with these findings, combined treatment yielded greater levels of FLT3- and syk- dephosphorylation compared to single agent treatment both when stroma was present and absent. When compared to untreated controls (11.6±0.7%), Annexin V/PI staining (n=3) showed that induction of apoptosis was increased with TAK (18.9±3.9%; p=NS), DEC (39.2±4.8%; p<.01), and greatest when the two drugs were combined (44.2±6.7%; p<.01) in the absence stoma. In the presence of stroma, single agent DEC, and when combined with TAK, increased the fraction of apoptotic cells by up to two fold whereas TAK did not induce any meaningful apoptosis. Consistent with the induction of cell death, cell cycle analysis demonstrated the greatest increase in subG0 phase when TAK and DEC were combined. Both in the presence and absence of stroma, autophagic flux was increased when M14 cells were treated with DEC, and when DEC was combined with TAK whereas no difference was observed with TAK alone. The combination of TAK and DEC led to changes consistent with differentiation, as shown by more abundant cytoplasm and nuclei with deep indentations, folds and lobulation compared to untreated cells. DEC treated cells also showed signs of differentiation although these changes were less marked. No evidence of differentiation was observed in M14 cells when treated with TAK alone.Conclusions: 1. Stroma conferred protective effects against all treatments. 2. TAK exerted dose dependent cytotoxicity against M14 cells in the nanomolar to low micromolar range in the absence and presence of stroma, respectively. 3. The cytotoxic effects of TAK were enhanced when TAK was combined with DEC. 4. The antileukemic effects of TAK combined with DEC are multifaceted and include inhibition of cell growth, induction of apoptosis, autophagy and differentiation. 5. The combination of TAK and DEC represents a potentially important novel approach for FLT3/ITD+ AML patients. 6. Clinical trials incorporating syk-inhibitors into standard AML treatment protocols are currently under way. DisclosuresDos Santos:Amgen: Employment.

Bone Marrow Stromal Cells Induce Bortezomib Resistance in Multiple Myeloma Cells through Downregulation of miRNA-101-3p Targeting Survivin

INTRODUCTIONIt is not yet fully understood how bone marrow microenvironment components especially bone marrow stromal cells (BMSCs) induce drug resistance in multiple myeloma (MM). This form of drug resistance has been suggested to pave the way for intrinsic (de novo) resistance to therapy in early stages of the disease and contribute to acquired drug resistance in the course of treatment. Hence, deciphering the molecular mechanisms involved in induction of above resistance will help identify potential therapeutic targets in MM combined treatments. Our previous work showed that BMSCs (normal and MM patient-derived) induced resistance to bortezomib (BTZ) compared with MM cells in the absence of stroma. This resistance was associated with modulation of a transcriptome in MM cells, including prominent upregulation of oncogenes c-FOS, BIRC5 (survivin) and CCND1. However; whether these oncogenes mediate BTZ resistance in the context of BMSCs through interaction with miRNAs is not known.METHODSHuman myeloma cell lines, 8226, U266 and MM.1s, were co-cultured with MM patient-derived BMSCs or an immortalized normal human line (HS-5) in the presence of 5nM BTZ for 24 h. MM cell monocultures treated with 5nM BTZ were used as controls. Co-cultures were then applied to magnetic cell separation (EasySep, Stem Cell Technologies) to isolate MM cells for downstream analyses (western blotting and qPCR). Total RNA including miRNAs was isolated from MM cell pellets (QIAGEN miRNeasy kit), cDNAs were synthesized (QIAGEN miScript RT II kit) and applied to miScript miRNA PCR Array (SABioscience, MIHS-114ZA). After normalization of all extracted Ct values to 5 different housekeeping genes, fold changes in miRNA expression were analyzed in co-cultures compared to MM cell monocultures using the 2-ΔΔCt algorithm. Moreover, survivin gene was silenced in MM cells using Ambion® Silencer® Select siRNA and Lipofectamine RNAiMAX transfection reagent. Survivin-silenced cells were then seeded on BMSCs and exposed to BTZ. Percent apoptosis of gated CD138+ MM cells was determined using FACS. For our overexpression and 3'UTR reporter experiments, we transiently transfected MM cells with pre-miR-101-3p, scrambled miRNA or pEZX-3'UTR constructs using Endofectin reagent (all from GeneCopoeia).RESULTSBMSCs upregulated survivin gene / protein (a member of inhibitors of apoptosis family) and modulated an array of miRNAs in MM cells compared to MM cells in the absence of stroma. The more noticeably downregulated miRNAs were hsa-miR-101-3p, hsa-miR-29b-3p, hsa-miR-32-5p, hsa-miR-16-5p (4-30 fold) and highly upregulated ones included hsa-miR-221-3p, hsa-miR-409-3p, hsa-miR-193a-5p, hsa-miR-125a-5p (80-330 fold). We focused on miRNA-101-3p as it showed the highest level of downregulation (30 fold) and has been shown to function as an important tumor suppressor in other malignancies. Real time RT-PCR confirmed downregulation of miRNA-101-3p. Moreover, microRNA Data Integration Portal (mirDIP) identified miRNA-101-3p as a putative target for survivin and Luciferase activity assays confirmed binding of miRNA-101-3p to 3'UTR of survivin. In addition, overexpression of miRNA-101-3p downregulated survivin and sensitized MM cells to BTZ-induced apoptosis. Furthermore, silencing of survivin upregulated miRNA-101-3p and increased BTZ-induced apoptosis in MM cell lines both in the absence of BMSCs (Apoptosis range in BTZ-treated conditions: 57.65% ± 4.91 and 28.66% ± 0.78 for si-survivin and scrambled control, respectively, p<0.05) and in the presence of BMSCs (41.23% ± 1.43 and 14.8% ± 0.66, for si-survivin and scrambled control, respectively, p<0.05).CONCLUSIONOur results indicate that BMSCs downregulated miRNA-101-3p and upregulated survivin in MM cells compared to MM cells in the absence of stroma. Silencing of survivin or overexpression of miRNA-101-3p sensitized MM cells to BTZ in the presence of BMSCs. These findings suggest that miRNA-101-3p mediates BTZ response of MM cells in the presence of BMSCs by targeting survivin and disclose a role of survivin-miRNA-101-3p axis in regulation of BMSCs-induced BTZ resistance in MM cells, thus provide a rationale to further investigate the anti-myeloma activity of miRNA-101-3p in combination with BTZ as a potential novel therapeutic strategy in MM. DisclosuresNo relevant conflicts of interest to declare.

Treatment with G-CSF reduces acute myeloid leukemia blast viability in the presence of bone marrow stroma.

BackgroundThe resulting clinical impact of the combined use of G-CSF with chemotherapy as a chemosensitizing strategy for treatment of acute myeloid leukemia (AML) patients is still controversial. In this study, the effect of ex vivo treatment with G-CSF on AML primary blasts was studied.MethodsPeripheral blood mononuclear cells from AML patients were treated with G-CSF at increasing doses, alone or in co-culture with HS-5 stromal cells. Cell viability and surface phenotype was determined by flow cytometry 72 h after treatment. For clonogenicity assays, AML primary samples were treated for 18 h with G-CSF at increasing concentrations and cultured in methyl-cellulose for 14 days. Colonies were counted based on cellularity and morphology criteria.ResultsThe presence of G-CSF reduced the overall viability of AML cells co-cultured with bone marrow stroma; whereas, in absence of stroma, a negligible effect was observed. Moreover, clonogenic capacity of AML cells was significantly reduced upon treatment with G-CSF. Interestingly, reduction in the AML clonogenic capacity correlated with the sensitivity to chemotherapy observed in vivo.ConclusionsThese ex vivo results would provide a biological basis to data available from studies showing a clinical benefit with the use of G-CSF as a priming agent in patients with a chemosensitive AML and would support implementation of further studies exploring new strategies of chemotherapy priming in AML.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-015-0272-3) contains supplementary material, which is available to authorized users.

Phosphoflow-Based Evaluation of Mek Inhibitors as Small-Molecule Therapeutics for B-Cell Precursor Acute Lymphoblastic Leukemia

Upstream mutations that lead to constitutive activation of Erk in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are relatively common. In the era of personalized medicine, flow cytometry could be used as a rapid method for selection of optimal therapies, which may include drugs that target the Erk pathway. Here, we evaluated the utility of phospho-flow, compared to Western blotting, to monitor Erk pathway activation and its inhibition by targeted Mek kinase inhibitors in human BCP ALL. Because the Erk pathway is not only activated endogenously, by mutations, but also by normal extracellular stimulation through stromal contact and serum growth factors, we compared Erk activation ex vivo in ALL cells in the presence and absence of stroma and serum. Phospho-flow was able to readily detect changes in the pool of pErk1/2 that had been generated by normal microenvironmental stimuli in patient-derived BCP-ALL cells passaged in NSG mice, in viably frozen primary patient samples, and in fresh patient samples. Treatment with the Mek1/2 inhibitor selumetinib resulted in a rapid, complete and persistent reduction of microenvironment-generated pErk1/2. Imaging flow cytometry confirmed reduction of nuclear pErk1/2 upon selumetinib treatment. An ALL relapsing with an activating KRasG12V mutation contained higher endogenous as well as serum/stromal-stimulated levels of pErk1/2 than the matched diagnosis sample which lacked the mutation, but selumetinib treatment reduced pErk1/2 to the same level in both samples. Selumetinib and trametinib as Mek inhibitors were mainly cytostatic, but combined treatment with the PI3K∂ inhibitor CAL101 increased cytotoxicity. Thus phospho-flow cytometry could be used as a platform for rapid, individualized in vitro drug sensitivity assessment for leukemia patients at the time of diagnosis.

Functional mapping of multiple myeloma kinome with library of small molecule inhibitors

The functional role of individual kinases in multiple myeloma (MM) cells has been primarily interrogated using RNAi approaches, which are mechanistically dissimilar to the pharmacological action of small molecule inhibitors used in the clinic; and in stroma-free cultures, which don’t account for stroma-induced signaling events. To address these dissimilarities, we performed functional mapping of the kinome in 16 MM cell lines, cultured in the presence vs. absence of stromal cells, using a panel of 273 kinase inhibitors (100nM, 24-72 h exposure) targeting a total of 43 known primary oncogenic targets. The cell-autonomous activity of the various inhibitors was broadly potent for Aurora, PLK, and mTORC1/2 inhibitors (consistent with high proliferative rates of MM cell lines in vitro); modest to minimal for selective PDK1, PI3K (excluding those that also inhibit mTOR), and Akt inhibitors; virtually none for selective inhibitors of c-met, ALK, EGFR, HER2, c-kit, PDGFR, VEGFR, Flt3, FAK, Syk, Src, and BTK (even in cell lines with detectable transcripts for the respective kinases). We also observed modest activity for FGFR3 inhibitors against FGFR3expressing cell lines; and none for BRAF inhibitors in our BRAF wild-type cell lines (some of which had increased proliferation upon treatment; consistent with known adaptive activation of signaling through C-RAF). Testing 8 different MEK1/2 inhibitors identified lines with low, intermediate vs. high response; the latter being KRAS-mutant and BRAF-wild-type cells with high baseline p-ERK levels (e.g. due to MEK2-activating mutation in one cell line). Importantly, comparison of results obtained in the presence vs. absence of stroma, indicate heterogeneous effect of bone marrow stromal cells on MM cell response to inhibitors across different targets and cell lines, including stroma-induced resistance to the same kinase inhibitor for some cell lines vs. sensitization for others. Our studies demonstrate the feasibility of functional annotations of the kinome in MM using libraries of small-molecule kinase inhibitors in phenotypic assays against panels of cell lines. Our results provide insight into the relative role of different classes of kinases in MM, and how the genetic (e.g. MEK2 or BRAF mutation status) and microenvironmental context can influence the role of these kinases and possible future individualized administration of inhibitors against these kinases in MM. BP-032

SHH Inhibitor PF-0449913 in Vitro Remodels Leukemic Niche into HSC Supportive Niche

BackgroundAcute myelogenous leukemia (AML) is a highly lethal disease with only 20% of 5 years survival. Most AML patients experience relapse usually leading to death. Progression to therapy resistant AML is driven by leukemia stem cells (LSC) harboring enhanced survival, dormancy and self-renewal capacity in supportive niches. Growing evidence indicates that in the development of myeloid malignancies deregulation of stem cells activity is as important as deregulation of the BM microenvironment. Sonic hedgehog (SHH) pathway molecules in general and SMO in particular are well known to maintain the growth of cancer cells. However, it’s role in leukemia niche-stem cell interaction is not well defined. Therefore, here we investigate: 1/ how the small molecule, SMO antagonist, PF-0449913 impacts the AML BM microenvironment and 2/ how in turn, changes in the activity of AML BM niche cells contribute to its remodeling into HSC supportive niche.MethodsCD34- cell from bone marrow patients undergoing hip replacement surgery (normal BM) as well as AML bone marrow were utilized for the development of the primary human stromal monolayers. We investigated stromal cultures of primary normal BM (NBM, n=6) and AML (n=6). As a control human normal bone marrow stromal cell line HS-5 was used. Then human CD34+ cells were selected from AML primary samples (n=6). As a normal control, CD34+ cells from cord blood (CB, n=5), or NBM (n=5) were utilized for the co-culture experiments for up to 9 weeks and then plated in survival and self-renewal assays. PF-0449913 was added to stromal co-cultures or, as a pre-treatment, directly to stromal monolayers.ResultsIn order to examine the role of SMO regulation in LSC generation and maintenance, primary AML (n=6) or cord blood (n=3) CD34+ cells were co-cultures with normal BM (n=3) or AML (n=3) stroma in the presence of novel small molecule inhibitor (PF-0449913) for 2 weeks. It significantly reduced LSC survival and self-renewal, while spared HSC. These effects were not observed in the absence of stroma. Importantly, pre-treatment of the AML stroma with PF-0449913 for 1 week prior- to co-culture also resulted in LSC’s inhibition of survival and self-renewal.AML- and normal BM-derived stroma differ in their ability to support HSC and LSC: LSC (n=6) were capable to self-renew after 9 weeks of co-culture with both normal and AML stroma, while cord blood (n=4) as well as normal BM (n=5) HSC lost their self-renewal potential after only 2 weeks of co-culture with the AML stroma. PF-0449913 induced changes in AML stroma. which was previously unsupportive to HSC survival, it became compliant : self-renewal of HSC increased more than 3 times. It reversed also the inhibitory effect of AML stroma-derived conditioned media (CM).Pre-treatment of the HS-5 cells with CM from AML stroma for 4 weeks prior to co-culture experiments led to significant inhibition of the cord blood (n=3) and NBM (n=3) HSC survival and self-renewal. However, in CM obtained from AML stroma treated with PF-0449913 that effect was reversed.Combined treatment of AML cells (n=6) with PF-0449913 and de-methylating agent Vidaza (5-azacytodine) or CXCR4 antagonist AMD3100 resulted in even greater inhibition of LSC survival and self-renewal. However, Vidaza did not mediate changes in AML stroma.ConclusionsTogether these data indicate that, while both conditioned media and co-culture with AML stroma impaired HSC survival and self-renewal, co-culture conditions resulted in a greater reduction in survival and self-renewal capacity, implicating that cell-cell contact or unstable secreted factors exacerbate the effects. That suggests that microenvironmental cues play a key role in regulating normal HSC versus LSC survival and maintenance, and leukemic stroma exibit severely compromised ability to maintain normal HSCs, but effectively support LSCs. PF-0449913treatment of AML induces not only eradication of LSC, but also mediate changes in AML stroma, supporting HSC generation and maintenance.Simultaneous targeting of both AML niche and LSC could represent a novel avenue for treatment of AML patients. DisclosuresNo relevant conflicts of interest to declare.

Regulation of Self-Renewal through Modulation of Let-7 Stem Cell Regulatory Family of microRNAs in Chronic Myeloid Leukemia Stem Cells

IntroductionChronic myeloid leukemia (CML) was one of the first malignancies shown to be initiated in hematopoietic stem cells by the BCR-ABL1 oncogene and sustained in blast crisis (BC) by progenitor cells that co-opt stem cell properties and behave like leukemia stem cells (BC LSCs). The BCR-ABL fusion oncogene encodes a constitutively active tyrosine kinase BCR-ABL. Although tyrosine kinase inhibitor (TKI) therapy targeting BCR-ABL suppresses CML during the chronic phase (CP), progenitors undergo expansion as a consequence of subsequent genetic and epigenetic alterations that fuel blast crisis transformation, BC LSC generation and TKI resistance. Self-renewing human BC LSCs harbor increased expression of Inflammation responsive adenosine deaminase acting on RNA (ADAR1), which can alter transcript as well as microRNA (miRNA) maturation, splicing and translation by Adenosine (A)-to-Inosine (I) editing of double stranded RNA. miRNAs are a family of small non-coding RNA molecules that regulate gene expression at a post-transcriptional level by inhibiting protein translation and/or reducing mRNA stability. Eukaryotic cells employ miRNAs in diverse biological processes including cell proliferation, differentiation, pluripotency and self-renewal. The stem cell pluripotency RNA binding protein LIN28B plays critical roles in BC transformation of CML. In this study we sought to characterize CML related-oncogenes, such as BCR-ABL, JAK2 and ADAR1, alone or in stromal co-culture in terms of their ability to regulate LSC self-renewal through modulation of let-7 /LIN28B stem cell transcriptional regulatory axis.MethodsMiRNAs were extracted from purified CD34+ cells derived from CP and BC CML patient samples as well as cord blood by RNeasy microKit (QIAGEN) and let-7 expression was evaluated by qRT-PCR using miScript Primer assay (QIAGEN). CD34+ cord blood (n=3) were transduced with lentiviral human BCR-ABL, JAK2, let-7a, wild type ADAR1 and ADAR1 mutant, which lacks a functional deaminase domain. Then, 72 hours after transduction, lentivirally transduced cells were plated on irradiated SL/M2 cells. After 5 days of culture, cells were collected for RNA and microRNA extraction. Transduction efficiency and LIN28B levels were evaluated by qRT-PCR and let-7 expression was quantified by qRT-PCR using miScript primer assay. Hematopoietic Progenitor and Replating assaywere performed on lenti-let-7a-overexpressing CB cells to assess differentiation, survival and self-renewal capacity.ResultsLentiviral overexpression of human BCR-ABL in CD34+ CB did not induce any significant change in let-7 family members and LIN28B expression in absence of stromal co-culture. However, stromal co-culture of BCR-ABL overexpressing CB led to the significant downregulation of members of the let-7 family as well as to upregulation of their target gene LIN28B, thus suggesting that extrinsic microenvironmental cues are necessary for modulating let-7 family levels in presence of BCR-ABL. Notably, qRT-PCR of CB transduced with JAK2 showed significant upregulation of ADAR1 in the absence of stroma, thus suggesting that JAK2 might be a mediator of inflammatory cytokine-driven ADAR1 activation. Lentiviral overexpression of both human JAK2 and ADAR1 significantly reduced the expression of let-7 family members and induced up-regulation of LIN28B. Interestingly, lentiviral overexpression of ADAR1 mutant did not induce any significant change in most let-7 family members. Finally, lentiviral overexpression of let-7a induced significant reduction in survival and self-renewal.ConclusionThese finding suggest that BCR-ABL requires extrinsic signals from the niche to modulate self-renewal of BC LSCs. Conversely, lentiviral JAK2 overexpression induces activation of aberrant RNA editing and subsequent reduction of let-7 family members in the absence of the niche. Interestingly, experiments with ADAR1 mutant, suggest that ADAR1 downregulates most of the let-7 family members in a RNA–editing dependent way manner. In summary these findings suggest a novel mechanism for BC LSC generation that may have utility in prognostication and selective LSCs targeting. DisclosuresJamieson:J&J: Research Funding; Sanofi: Research Funding, Travel Support, Travel Support Other; Roche: Research Funding.

Single luminal epithelial progenitors can generate prostate organoids in culture.

The intrinsic ability to display self-organizing morphogenetic properties in ex vivo culture may represent a general property of tissue stem cells. Here we show that single luminal stem/progenitor cells can generate prostate organoids in a three-dimensional culture system in the absence of stroma. Organoids generated from CARNs (castration-resistant Nkx3.1-expressing cells) or normal prostate epithelium exhibit tissue architecture containing luminal and basal cells, undergo long-term expansion in culture, and display functional androgen receptor signaling. Lineage-tracing demonstrates that luminal cells are favored for organoid formation, and generate basal cells in culture. Furthermore, tumor organoids can initiate from CARNs after oncogenic transformation, and from mouse models of prostate cancer, and can facilitate analyses of drug response. Finally, we provide evidence supporting the feasibility of organoid studies of human prostate tissue. Our studies underscore the progenitor properties of luminal cells, and identify in vitro approaches for studying prostate biology.

Intermediate-Risk Acute Lymphoblastic Leukemia (ALL) Patients with and without Relapse Differentially Depend on Survival Signals From Microenvironment

Abstract 752Risk stratification in the childhood ALL-BFM clinical trial has been based on molecular/cytogenetic markers and the in vivo response to treatment (blast reduction in peripheral blood after prednisone prephase, bone marrow clearance after induction treatment and minimal residual disease (MRD) after induction and induction consolidation). These criteria classify about 50% of ALL cases into standard-risk (SR) and high-risk (HR) groups. The intermediate-risk (IR) ALL group, showing moderate levels of MRD, provides the majority of relapses, but still lacks specific prognostic markers which could distinguish between those patients subsequently relapse and those remaining relapse-free (IR+r and IR-r). We established a bank of NOD/SCID ALL xenografts representing IR+r and IR-r subgroups (12 vs 13 patients) and characterized the samples in a broad series of functional assays. There was no difference in the sensitivity to drugs (dexamethasone, daunorubicin, L-asparaginase and vincristine) between the IR+r and IR-r groups, however, the rate of spontaneous apoptosis in vitro was significantly higher in the IR+r group than in the IR-r group (69 +/− 7% vs 39 +/− 6%, p=0.02). Given that in the presence of stromal cells the cell death level has been similar in both IR groups (9 +/− 5% vs 7 +/− 7%, p=0.8), these observations suggest that the IR+r ALL is more dependent on survival signals from microenvironment than IR-r ALL. In the NOD/SCID engraftment model, leukemic cells from IR+r patients engrafted more rapidly than cells from IR-r patients (93 +/− 12 days vs 170 +/− 24 days, p=0.006), pointing to a better in vivo cooperation between leukemia cells and microenvironment in the IR+r cases. In order to identify the underlying molecular pathways, gene expression changes induced in the presence and absence of stroma have been investigated at genome-wide level (Human Gene 1.0 ST Affymetrix array). Of the apoptosis-regulating genes, a caspase inhibitior from the IAP family, BIRC3, was found to be up-regulated significantly higher in the IR-r than in the IR+r group (3.7 +/− 1.1 vs 1.7 +/− 0.1-fold up-regulation, p=0.016). The adhesion molecule VCAM1 has been generally up-regulated by the co-incubation with stroma, with a tendency to a higher up-regulation in the IR-r (4.4 +/− 0.7-fold) than in the IR+r (2.7 +/− 1.0-fold) group. We further speculated that interaction with microenvironment is a mutual process which also implicates gene expression changes of the stroma. To this end, mouse stromal cells (MS5 cell line) were incubated with a series of ALL samples (n=3), purified by MACS sorting and investigated using Mouse Gene 1.0 ST Affymetrix arrays. The list of genes whose expression increased by at least 2-fold in all 3 cases (n=10), included the macrophage inflammatory peptide-1alpha (MIP1A/CCL3), a chemokine known to regulate chemotaxis of lymphocytes. Our data suggest that the cooperation of ALL with microenvironment may provide a promising approach to molecularly discriminate ALL cases with relapse and to identify potential relapse-associated targets within the IR-ALL group. Disclosures:No relevant conflicts of interest to declare.

BH3 Profiling Demonstrates That Restoration of Apoptotic Priming Contributes to Increased Sensitivity to PI3K Inhibition in Stroma-Exposed Chronic Lymphocytic Leukemia Cells

Abstract 974 We and others have previously found that CLL cells exposed to stroma are resistant to a wide variety of treatments. While this phenomenon is thought largely to be due to inhibition of apoptosis, this has not been directly tested. BH3 profiling was developed in our laboratory as a functional assay to assess the degree to which malignant cells are primed to undergo apoptosis via the mitochondrial pathway. BH3 profiling demonstrates that decreased priming for apoptosis is indeed associated with treatment resistance in CLL cells co-cultured with mouse fibroblast L cells. Here, we confirm these preliminary results, showing that CLL cells co-cultured with either primary human stroma or a human stromal cell line also exhibit decreased priming for apoptosis. We demonstrate that the delta-isoform specific PI3K inhibitor CAL-101 (GS1101) effectively kills stroma-exposed CLL cells and sensitizes them to treatment with other agents by releasing the cells from stroma and/or increasing their level of mitochondrial apoptotic priming. CLL cells were co-cultured in the presence or absence of drugs with either the human stromal cell line StromaNKTert, with nurse-like cells (NLC) grown from primary CLL patient samples, or with no stroma for 24 hours, as previously described (Burger et al., Blood, 2000). A calcein-based adhesion assay was used to assess CLL cell adhesion to stroma in the presence or absence of CAL-101 (GS1101). CLL cell apoptosis in response to various drug treatments was assessed by Annexin/PI analysis. BH3 profiling was performed by exposing gently permeabilized malignant CD19+ B cells from CLL patients to a panel of BH3-domain peptides, and mitochondrial apoptotic priming was quantified by JC-1-based FACS to assess mitchondrial outer membrane permeabilization, as previously described (Ryan et al., PNAS 2010). Stroma-exposed CLL cells were highly resistant to the purine analog fludarabine and the BH3-mimetic ABT-263. CAL-101 (GS1101) was able to partially overcome this resistance, and the combination of CAL-101 (GS1101) and fludarabine led to a marked increase in sensitivity of stroma-exposed CLL cells. To evaluate the underlying mechanism of increased CLL cell sensitivity in the presence of CAL-101 (GS1101), we labelled CLL cells with calcein, and co-cultured them with the StromaNKTert cell line. In short term culture (1 hour), CLL cells were highly adherent to stroma, and were significantly less adherent in the presence of CAL-101 (GS1101). After longer co-culture (24 hours), calcein-labelled CLL cells were released from stroma within one hour of the addition of CAL-101 (GS1101). To evaluate whether the release of CLL cells from stroma affected their apoptotic priming, we performed BH3 profiling on CLL cells co-cultured in the presence or absence of stroma. CLL cells cultured without stroma were significantly more primed for apoptosis than CLL cells co-cultured with either the StromaNKTert cell line or primary human NLC. The addition of CAL-101 (GS1101), with or without fludarabine, to CLL cells in culture with stroma, led to increased apoptotic priming compared to CLL cells in culture that were untreated or treated with fludarabine alone. Similar alterations in apoptotic priming were observed when CLL cells were co-cultured with CAL-101 (GS1101) with or without ABT-263. Overall, these results suggest that the sensitivity of stroma-exposed CLL cells to CAL-101 (GS1101) may be related to the increased mitochondrial apoptotic priming that is facilitated by release of these cells from the stroma. These data may partially explain the clinical observation that, unlike traditional chemotherapy agents, CAL-101 (GS1101) has been efficacious in CLL despite causing an increase in the white blood cell count. Disclosures: Lannutti: Gilead Sciences: Employment. Letai:Eutropics Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees.

2.26 Clopidogrel (Plavix) Induces In Vivo Reduction of Normal B Cells and In Vitro Apoptosis of B-Cell Chronic Lymphocytic Leukemia Cells

using human stroma. We then explore the hypothesis that the deltaisoform specific PI3K inhibitor CAL-101 is able to both effectively kill stroma-exposed CLL cells and sensitize them to treatment with other agents, by releasing the cells from stroma and/or increasing their level of mitochondrial apoptotic priming. Methods: CLL cells were co-cultured with or without drug treament for 24 hours in the absence of stroma and with either the human stromal cell line StromaNKTert or with nurse-like cells (NLC) grown from primary CLL patient samples as previously described. CLL cell adhesion to stroma in the presence or absence of CAL-101 was visualized using a Calcein-based adhesion assay, and quantified by fluorimetry. CLL cell viability was assessed by Annexin/PI analysis. BH3 profiling was performed by exposing malignant CD19 B cells from CLL patients to a panel of BH3-domain peptides, and mitochondrial apoptotic priming was quantified by JC-1-based FACS to assess mitchondrial outer membrane permeabilization, as previously described. Results: CLL cells exposed to human stroma were resistant to treatment with the purine analog fludarabine and the BH3-mimetic ABT-263. CAL-101 was able to partially overcome this resistance, and the combination of CAL-101 and fludarabine led to a marked increase in sensitivity of stroma-exposed CLL cells. To investigate the mechanism of this increased sensitivity in the presence of CAL-101, we labelled CLL cells with calcein, and co-cultured them with the StromaNKTert cell line. We found that the CLL cells were highly adherent to stroma after only one hour of co-culture, and were significantly less adherent in the presence of CAL-101. Also, a significant number of calcein-labelled CLL cells co-cultured with stroma for 24 hours were released from stroma within one hour after the addition of CAL-101. To determine the effect of the release of CLL cells from stroma on apoptotic priming, we performed BH3 profiling on CLL cells co-cultured in the presence or absence of stroma. CLL cells cultured in the absence of stroma were significantly more primed for apoptosis than CLL cells co-cultured with either the StromaNKTert cell line or primary human NLC. The addition of CAL-101, with or without fludarabine, to CLL cells in culture with stroma, led to increased apoptotic priming compared to CLL cells in culture that were untreated or treated with fludarabine alone. Conclusions: Taken together, these results suggest that the sensitivity of stromaexposed CLL cells to CAL-101 may be related to the increased mitochondrial apoptotic priming that is facilitated by release of these cells from the stroma. These data may partially explain the clinical observation that, unlike traditional chemotherapy agents, CAL-101 has been efficacious in CLL despite causing an increase in the white blood cell count.

βig-h3 Regulates Differentiation and Survival of Human Hematopoietic Stem/Progenitor Cells.

AbstractAbstract 2591Adult hematopoietic stem cells (HSC) reside in dedicated niches in the bone marrow (BM). Within this specialized microenvironment, the various interactions of HSC with adhesion molecules on neighbouring cells and extracellular matrix (ECM) components are critical for the maintenance of the HSC population and the concomitant development of the distinct blood cell lineages. Comparative gene-expression profiling of purified HSC identified ECM proteins that are differentially expressed in homeostatic and regenerative conditions. The ECM protein βig-h3 was one of the proteins upregulated in regenerative conditions. Therefore, we characterized the role of βig-h3 in the regulation of HSC self-renewal and differentiation.A comparison between human CD34+ hematopoietic stem/progenitor cells (HSPC) isolated from BM, mobilized peripheral blood (MPB) and umbilical cord blood (UCB), revealed the highest βig-h3 expression in BM-HSPC (3.9-fold increased compared to MPB, 1.7-fold increased compared to CB), which may implie a role for βig-h3 in retaining HSC in the BM.To examine the functional relevance of βig-h3 in HSC, we first increased βig-h3 expression by transducing human HSPC with a lentiviral βig-h3-SIN-GFP expression vector or a control SIN-GFP vector. Over-expression of βig-h3 (80-fold) in HSPC decreased colony-forming-unit-granulocyte-monocyte (CFU-GM) formation from 130 (SEM=47) to 73 (SEM=19, n=3) CFU-GM per 500 plated CD34+ cells, while megakaryopoiesis was accelerated and the number of mature megakaryocytic cells increased from 16% (SEM=6%) to 30% (SEM=7%, n=4) at day 14 of culture. Ectopic expression of βig-h3 did not affect differentiation along the erythroid or granulopoietic lineage. The development of megakaryocytes at the cost of pluripotent CFU-GM suggests that βig-h3 drives differentiation.In addition, βig-h3 expression in HSPC was reduced by two different short-hairpin-RNAs (shRNA) expressed from lentiviral vectors, which resulted in decreased proliferation (from 19.6- to 5.8-fold per input cell at day 13) and increased apoptosis (from 13.5% to 25.3% at day 13) in liquid HSPC cultures, as analyzed by Annexin V staining. Similarly, knock-down of βig-h3 in various cell lines also resulted in a decreased proliferation and increased apoptosis. Knock-down of βig-h3 in primary HSPC dramatically reduced CFU-GM from 73 (SEM=8.7) to 31 (SEM=14.4, n=6) CFU-GM per 500 CD34+ cells plated, and reduced colony-forming-unit-erythrocyte (CFU-E) formation from 30 (SEM=6.5) to 9 (SEM=1.6, n=4) CFU-E per 500 CD34+ cells plated. This can be explained by increased apoptosis of βig-h3 knock-down cells. Notably, co-culture of βig-h3 knock-down HSPC with stromal feeder cells, known to express high levels of βig-h3, showed no difference compared to control HSPC in cobblestone area formation within two weeks, indicating that stromal cells can counteract apoptosis in βig-h3 knock-down cells. Remarkably, long-term-culture CFU-GM (LTC-CFU) formation of HSPC that were co-cultured with stromal cells during two weeks, was even significantly increased (1.9-fold, n=2) in βig-h3 knock-down cells, indicating that decreased endogenous levels of βig-h3 stimulates the maintenance or expansion of HSPC on stroma.In conclusion, ectopic expression of βig-h3 decreased CFU-GM in HSPC and accelerated differentiation towards megakaryocytes, suggesting that βig-h3 might drive lineage commitment of HSC. Conversely, knock-down of βig-h3 in HSPC stimulated LTC-CFU formation, indicating that decreased βig-h3 levels in HSPC maintain their undifferentiated state. In absence of stroma, however, knock-down of βig-h3 induces apoptosis, indicating βig-h3 as an essential survival factor, which expression levels regulate differentiation and maintenance of HSC. Disclosures:No relevant conflicts of interest to declare.

How to Overcome Myeloma Stem Cell Resistance to Therapy - Targeting the Stem Cell Niche

AbstractAbstract SCI-6The concept of cancer stem cells has attracted again intense research interest, as the drug resistance attributed to this infrequent subpopulation of tumor cells could explain how patients can relapse even after prolonged complete clinical, biochemical, radiologic or even molecular remissions. In multiple myeloma (MM), several aspects of the cancer stem cell concept remain to be elucidated, including the potential heterogeneity of this cell subpopulation or whether CD138+ expression is incompatible or not with a MM stem cell. As these questions are being resolved, emerging data highlight that the drug resistance of MM cells with clonogenic/stem cell-like features is heavily influenced by interactions with non-malignant accessory cells of the local microenvironment, including bone marrow stromal cells (BMSCs). Indeed, transcriptional signatures of “stemness”, as identified in normal stem cells or cancer stem cells from other neoplasias, are detected in the bulk population of MM cell lines and are upregulated after MM cell interaction with BMSCs. MM cell lines and primary tumor cells contain subpopulations with clonogenic potential, such as the side population (SP) cells. SP cells, detected by low intracellular accumulation of Hoechst 33342 dye (in contrast to the tumor's “main population” (MP), are considered an enriched source of tumor-initiating cells in diverse neoplasias and were detected among CD138− CD20+ CD27+ clonogenic cells in primary MM samples. Interaction with BMSCs increases the viability of SP cells and their percentage within the MM cell population. While interaction with BMSCs or other accessory cells of the microenvironmental niche suppresses the anti-MM activity of glucocorticoids, conventional chemotherapeutics and certain investigational agents, other agents (e.g. immunomodulatory thalidomide derivatives (IMIDs), such as lenalidomide) have increased activity against MM SP cells in the context of this tumor-microenvironment interaction. These observations suggest that MM cells with stem cell-like features exhibit functional plasticity depending on which specific microenvironmental niche they interact with. The Hedgehog, Wnt and Notch pathways, as well as regulators of chromatin remodeling, e.g. histone demethylases, have emerged as putative links between drug resistance, “cancer stemness” and how these functional outcomes are modulated by the local microenvironment in MM. These pathways, as well as embryonic stem cell-associated antigens (e.g. SOX2), represent intriguing targets for investigational therapies. However, clinical translation of such treatments has notable challenges, as conventional criteria for response assessment may not accurately reflect the treatment's impact on clonogenic tumor cells. Progression-free survival is considered a more appropriate endpoint for cancer stem-cell targeting agents, its assessment, however, may be confounded without concomitant treatment that suppresses the bulk of the tumor. Consequently, candidate cancer stem cell-targeting agents may have to be evaluated in combination with regimens (including lenalidomide-bortezomib-Dex (RVD) or other combinations built around the therapeutic “backbone” of proteasome inhibition and IMIDs) which potently target the bulk MM cell population and induce high rates of complete/near complete responses. Further improvements of this dual targeting of clonogenic and bulk tumor cells may be facilitated by recently developed high-throughput platforms (e.g. compartment-specific bioluminescence imaging, CS-BLI) which screen, in the presence vs. absence of stroma or other accessory cells, large numbers of anti-tumor agents and combinations thereof against the bulk tumor cell population or its clonogenic compartments. These new platforms will inform the rational design of regimens that will hopefully improve the long-term outcome of MM patients by suppressing a clonogenic/stem cell-like tumor compartment and circumventing microenvironment-dependent drug resistance. Disclosures:Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.

Stromal Rescue of Drug Exposed CLL Cells Can Be Overcome by the Green Tea Extract Epigallocatechin 3 Gallate

AbstractAbstract 3610 PurposeChronic Lymphocytic Leukemia (CLL) is incurable with conventional chemotherapy treatments. Nurturing of leukemic CLL B cells by marrow microenvironment appears to be one important method of chemoresistance (NEJM 2005).Epigallocatechin 3 gallate (EGCG), the major catechin in green tea, induces caspase-dependent death CLL B-cells and down regulates anti-apoptotic proteins (Mcl-1; XIAP) critical for enhanced apoptosis resistance of CLL B-cells (Blood 2004). In Phase I and II clinical testing, we found EGCG induced a decline in absolute lymphocyte count and/or lymphadenopathy in the majority of Rai stage 0-II CLL patients (JCO 2009; ASCO 2010).In these phase I/II studies, trough plasma EGCG levels as high as 8 mM were achieved. Since it does not cause myelosuppression, EGCG has the potential to be combined with other chemotherapeutic agents used for CLL. We previously reported that pharmacologically achievable plasma levels of EGCG have an additive or synergistic effect when combined with fludarabine (F), chlorambucil (C), and FC in the majority of patients (ASH 2009). In this study, we evaluated i) the effect of pharmacologically achievable doses of EGCG when combined with bendamustine (B) and ii) the ability of pharmacologically achievable doses of EGCG to overcome in vitro stromal rescue of CLL B-cells exposed to B or FC. MethodsWe first evaluated the effects of EGCG combined with B to establish if this partnership would be additive or synergistic. Primary CLL cells were treated with B (12.5-100 mM) with or without 4 uM EGCG. After 48 hours cells were stained with annexin/PI, and viability analyzed by flow cytometry. Data were analyzed using the CalcuSyn program (Chou & Talay method) to determine the combination index (indicating antagonistic, additive or synergistic effects). Next, we evaluated the ability of 4 mM EGCG to overcome stromal rescue of CLL cells treated with B or FC. To eliminate the cytotoxic effects of FC and B on stromal cells, we treated CLL cells with FC or B for 24 hours, washed with PBS, and then cultured these drug exposed cells directly on the HS-5 human stromal cell line with or without 4 mM EGCG for 48 hours (FC) or 24 hours (B). Cells were harvested and viability of CD19+ cells was assessed by annexin/PI staining. To specifically evaluate the ability of 4 mM EGCG to overcome stromal protection, co-culture samples in which no stromal protection was observed were excluded. Similar experiments were conducted using primary bone marrow mesenchymal stromal cells (MSC) from CLL patients (Leuk Res, 2007). In these experiments the fludarabine/chlorambucil combination was used as an approximation of fludarabine/cyclophosphamide since the active form of cyclophosphamide (4-hydroxycyclophosphamide) rapidly breaks down in aqueous buffer and is not readily available for in vitro use and. ResultsWe previously observed additive or synergistic FC and EGCG interactions in a majority of patient samples (ASH 2009). 4 mM EGCG was less consistently beneficial when combined with increasing doses of B in the absence of stroma. Synergistic or mixed synergistic/additive effect was seen in 5 patients, mixed additive/antagonistic effects in 3, and antagonistic effects in 2. Given previously observed synergy with FC and additive/synergistic interactions with at least some B treated samples, we next explored the effects of EGCG on stromal cell rescue. Co-culture with HS-5 stromal cells significantly reduced both B and FC mediated cell death. However the addition of 4 μM EGCG significantly attenuated stromal cell rescue of CLL B-cells exposed to B and FC therapy (Fig. 1A and 1B respectively). Remarkably in the case of FC, 4 mM EGCG almost completely abrogated stromal rescue. Experiments using primary bone marrow MSC cultures from CLL patients yields similar results (n=3, data not shown). [Display omitted] [Display omitted] ConclusionsPharmacologically achievable doses of EGCG appear to reduce stromal rescue of CLL cells treated with B or combinations of purine nucleoside analog/alkylating agents. This occurs in the absence of detectable stromal cell kill by EGCG. These findings, coupled with the favorable toxicity profile and lack of myelosuppression in phase I/II trials of EGCG make it an attractive agent to test in combination with bendamustine or purine analogue/alkylating agent chemo-immunotherapy for patients with CLL. Disclosures:Shanafelt:Polyphenon Pharma: Research Funding.

Papanicolaou tests associated with cervical mucosal endometriosis: An analysis of cellular features and comparison to endocervical adenocarcinoma in situ

Endometrium directly sampled from endocervical mucosal endometriosis can mimic endocervical adenocarcinoma in situ (AIS) in Papanicolaou (Pap) tests. We analyzed a series of Pap tests to investigate the cellular features of mucosal endometriosis and to assess the utility of stroma and apoptotic bodies in the differential diagnosis with AIS. Pap test samples from patients known to have endocervical mucosal endometriosis were compared with samples containing AIS. Pap tests from patients with mucosal endometriosis had lesional cells in 13 (62%) cases which includes glandular and stromal cells (10 cases), stroma only (two cases), and glandular cells only (one case). Three (23%) cases had gland-stromal aggregates. Three (23%) cases had mitotic figures and two (15%) had apoptotic bodies. By comparison, only one (8%) AIS case had endometrial-type stroma. Seven (58%) AIS cases had apoptotic bodies and three (25%) had mitotic figures. We conclude that Pap tests from patients with mucosal endometriosis usually (62%) have lesional cells. These lesional cells almost always include stroma, which is useful in the differential diagnosis with AIS. We identified stroma significantly more often in endometriosis cases (92%) than in AIS cases (8%). Pathologists should look for endometrial stroma when considering an interpretation of directly sampled endometrium. In the absence of stroma, AIS should be considered.

A Functional Receptor for B-Cell–Activating Factor Is Expressed on Human Acute Lymphoblastic Leukemias

B-lineage acute lymphoblastic leukemia (ALL) arises by transformation of a progenitor (pre-B) cell. Cure rates in adults remain low and treatment is complicated by support provided by the microenvironment to the leukemic cells, indicating an urgent need to better understand the factors that promote their survival. B-cell-activating factor (BAFF) and its receptor BAFF-R are important for survival and growth of mature normal and malignant B cells but are not expressed on pre-B cells. Unexpectedly, all cells in the primary Philadelphia chromosome (Ph)-positive and Ph-negative ALL samples tested were positive for high BAFF-R cell surface expression. BAFF-R was fully competent to bind BAFF, and stimulation of the receptor activated both the classic and the noncanonical NF-kappaB pathways. Recombinant BAFF supported survival of the ALL cells in the absence of stroma, and it significantly attenuated the rate of apoptosis caused by exposure to nilotinib, a drug used therapeutically to treat Ph-positive ALLs. Surprisingly, BAFF mRNA and protein were also expressed in the same cells but BAFF was not shed into the medium. Our report is the first showing universal expression of BAFF-R by pre-B ALL cells and opens the possibility of blocking its function as an adjuvant therapeutic strategy.

Basal cells as stem cells of the mouse trachea and human airway epithelium

The pseudostratified epithelium of the mouse trachea and human airways contains a population of basal cells expressing Trp-63 (p63) and cytokeratins 5 (Krt5) and Krt14. Using a KRT5-CreER(T2) transgenic mouse line for lineage tracing, we show that basal cells generate differentiated cells during postnatal growth and in the adult during both steady state and epithelial repair. We have fractionated mouse basal cells by FACS and identified 627 genes preferentially expressed in a basal subpopulation vs. non-BCs. Analysis reveals potential mechanisms regulating basal cells and allows comparison with other epithelial stem cells. To study basal cell behaviors, we describe a simple in vitro clonal sphere-forming assay in which mouse basal cells self-renew and generate luminal cells, including differentiated ciliated cells, in the absence of stroma. The transcriptional profile identified 2 cell-surface markers, ITGA6 and NGFR, which can be used in combination to purify human lung basal cells by FACS. Like those from the mouse trachea, human airway basal cells both self-renew and generate luminal daughters in the sphere-forming assay.

Paracortical axillary sentinel lymph node ectopic breast tissue

Benign glandular inclusions in axillary lymph nodes are uncommon, and their presence in axillary sentinel lymph nodes is exceptionally rare. The possibility of over-staging due to misinterpretation of glandular inclusions as metastatic carcinoma is a concerning issue. We present a 54-year-old female with high grade ductal carcinoma in-situ undergoing simple mastectomy with sentinel lymph node biopsy. Permanent sections of the sentinel lymph node revealed scarce naked small glands without surrounding stroma scattered in the paracortex in the superficial level. Deeper levels showed glands spanning a much larger area (2 mm), with bland ducts and tubules separated by abundant stroma. The myoepithelial layer was visible and was immunohistochemically confirmed. A final diagnosis of benign ectopic breast tissue within an axillary sentinel lymph node was rendered. Previous studies described axillary sentinel lymph nodes with glandular inclusions separated by stroma or subcapsular in location. It has been suggested that paracortical location and absence of stroma are characteristics of metastasis. As demonstrated in our report, benign inclusions may be paracortical and lack surrounding stroma. We recommend that glandular inclusions should be a diagnostic consideration for cases in which paracortically located naked glands do not histologically resemble the corresponding primary tumor.