Serial Transplantation Studies Research Articles

A niche for axial stem cells - A cellular perspective in amniotes

The head-tail axis in birds and mammals develops from a growth zone in the tail-end, which contains the node. This growth zone then forms the tailbud. Labelling experiments have shown that while many cells leave the node and tailbud to contribute to axial (notochord, floorplate) and paraxial (somite) structures, some cells remain resident in the node and tailbud. Could these cells be resident axial stem cells? If so, do the node and tailbud represent an instructive stem cell niche that specifies and maintains these stem cells? Serial transplantation and single cell labelling studies support the existence of self-renewing stem cells and heterotopic transplantations suggest that the node can instruct such self-renewing behaviour. However, only single cell manipulations can reveal whether self-renewing behaviour occurs at the level of a cell population (asymmetric or symmetric cell divisions) or at the level of single cells (asymmetric divisions only). We combine data on resident cells in the node and tailbud and review it in the context of axial development in chick and mouse, summarising our current understanding of axial stem cells and their niche and highlighting future directions of interest.

The E3 Ligase Itch Is a Regulator of Bone Marrow Mesenchymal Stem Cells and the Hematopoietic Stem Cell Niche

Hematopoietic stem cells (HSCs) reside in a complex cellular microenvironment within the bone marrow. Bone marrow mesenchymal stromal/stem cells (BM-MSCs) have emerged as an important cellular component of this hematopoietic stem cell niche. BM-MSCs have been implicated in physiological hematopoiesis as well as in hematological malignancies. The intracellular regulatory network that governs BM-MSC homeostasis and their function within the HSC niche remains poorly understood. Posttranslational modification of proteins through ubiquitylation has been proven an integrative component in the regulation of quiescence, proliferation, self-renewal and differentiation of stem cells. We have previously demonstrated that the E3 ubiquitin ligase Itch is a negative regulator of HSC homeostasis and function (Rathinam et al., Nat.Immunol., 2011). Notably, patients suffering from a rare ITCH deficiency syndrome display morphologic abnormalities with dysmorphic features, suggesting a role for this E3 ligase within the skeletal tissue and mesenchymal lineage cells (Lohr et al., Am.J.Hum.Genet., 2010).In the present study, we identify Itch as a critical regulator of BM-MSCs. Itch-/- mice exhibit an at least two-fold reduction of MSCs (immunophenotypically identified as CD45-TER119-CD31-PDGFRα+Sca-1+CD90+) in the BM. Consistently, Colony Forming Unit-Fibroblasts (CFU-F) assays revealed a remarkable reduction of MSCs in the BM (p=0.0392). Moreover, in-vitro culture studies identified a hyperproliferative phenotype of Itch mutant MSCs. However, further studies revealed that Itch deficiency leads to reduced differentiation of MSCs into osteoblasts, both in-vivo and in-vitro, and therefore resulting in osteopenic bone phenotype. Reciprocal and Serial Bone marrow transplantation studies suggested an alteration of the niche for HSCs in Itch deficient BM. Interestingly, Itch-/- recipients exhibited a five-fold increase in their donor (wild-type) hematopoietic stem and progenitor cell compartment. Molecular and biochemical studies documented an increase of signals mediated by Notch in Itch deficient MSCs and co-localization of Itch and Notch-1 intracellular domain. Of note these findings gain importance, as Notch signaling has been previously implicated in the regulation of MSC differentiation into osteoblasts, as well as in the regulation of the HSC niche.In summary, our studies identified Itch as a positive regulator of BM-MSC homeostasis. Our results suggest that the Itch-Notch axis within the HSC niche may serve as a potential pharmaceutical target, especially in the setting of bone marrow transplantation in humans. Treatment of the transplant recipient with an Itch inhibitor may avoid graft failure or rescue poor hematopoietic recovery. DisclosuresSchünemann:German Academic Exchange Service: Other: scholarship; Boehringer Ingelheim Fonds: Other: travel grant.

Asrij/OCIAD1 suppresses CSN5-mediated p53 degradation and maintains mouse hematopoietic stem cell quiescence

AbstractInactivation of the tumor suppressor p53 is essential for unrestrained growth of cancers. However, only 11% of hematological malignancies have mutant p53. Mechanisms that cause wild-type p53 dysfunction and promote leukemia are inadequately deciphered. The stem cell protein Asrij/OCIAD1 is misexpressed in several human hematological malignancies and implicated in the p53 pathway and DNA damage response. However, Asrij function in vertebrate hematopoiesis remains unknown. We generated the first asrij null (knockout [KO]) mice and show that they are viable and fertile with no gross abnormalities. However, by 6 months, they exhibit increased peripheral blood cell counts, splenomegaly, and an expansion of bone marrow hematopoietic stem cells (HSCs) with higher myeloid output. HSCs lacking Asrij are less quiescent and more proliferative with higher repopulation potential as observed from serial transplantation studies. However, stressing KO mice with sublethal γ irradiation or multiple injections of 5-fluorouracil results in reduced survival and rapid depletion of hematopoietic stem/progenitor cells (HSPCs) by driving them into proliferative exhaustion. Molecular and biochemical analyses revealed increased polyubiquitinated protein levels, Akt/STAT5 activation and COP9 signalosome subunit 5 (CSN5)-mediated p53 ubiquitination, and degradation in KO HSPCs. Further, we show that Asrij sequesters CSN5 via its conserved OCIA domain, thereby preventing p53 degradation. In agreement, Nutlin-3 treatment of KO mice restored p53 levels and reduced high HSPC frequencies. Thus, we provide a new mouse model resembling myeloproliferative disease and identify a posttranslational regulator of wild-type p53 essential for maintaining HSC quiescence that could be a potential target for pharmacological intervention.

Attenuation of PKCδ enhances metabolic activity and promotes expansion of blood progenitors

A finely tuned balance of self‐renewal, differentiation, proliferation, and survival governs the pool size and regenerative capacity of blood‐forming hematopoietic stem and progenitor cells (HSPCs). Here, we report that protein kinase C delta (PKCδ) is a critical regulator of adult HSPC number and function that couples the proliferative and metabolic activities of HSPCs. PKCδ‐deficient mice showed a pronounced increase in HSPC numbers, increased competence in reconstituting lethally irradiated recipients, enhanced long‐term competitive advantage in serial transplantation studies, and an augmented HSPC recovery during stress. PKCδ‐deficient HSPCs also showed accelerated proliferation and reduced apoptosis, but did not exhaust in serial transplant assays or induce leukemia. Using inducible knockout and transplantation models, we further found that PKCδ acts in a hematopoietic cell‐intrinsic manner to restrict HSPC number and bone marrow regenerative function. Mechanistically, PKCδ regulates HSPC energy metabolism and coordinately governs multiple regulators within signaling pathways implicated in HSPC homeostasis. Together, these data identify PKCδ as a critical regulator of HSPC signaling and metabolism that acts to limit HSPC expansion in response to physiological and regenerative demands.

Identification of Human Cutaneous Squamous Cell Carcinoma Cancer Stem Cells.

Epithelia are under constant threat from environmental carcinogens and none more so than squamous epithelia, which form the outermost linings of our bodies. Hence malignancies of squamous epithelia are collectively the most common cancer type and with the highest mortality, despite a constant cell turnover and only relatively rare long-lived adult tissue stem cells. Genetic analysis from SCC whole genome sequencing reveals commonality in mutated genes, despite various etiological factors. Most SCC types have been shown to exhibit hierarchical growth, in which a high frequency of cancer stem cells is associated with poor prognosis. For human cutaneous SCC (cSCC), we have shown that cancer stem cells express CD133 and that this population can recreate tumor heterogeneity in a novel in vivo model. CD133+ cSCC cells is small subset of tumor cells (~1%) in the outer layer of cSCC that are highly enriched for tumor-initiating capacity (TIC) (~1/400) compared to unsorted cSCC cells (~1/106). Xenografts of CD133+ cSCC recreated the original cSCC tumor histology and organizational hierarchy, while CD133- cells did not. Only CD133+ cells demonstrated the capacity for self-renewal in serial transplantation studies. Hence, cSCC has the potential to be the ideal model in which to study SCC biology.

ZFP521 regulates murine hematopoietic stem cell function and facilitates MLL-AF9 leukemogenesis in mouse and human cells

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

Abstract 1911: R-SPONDIN3 antagonism sensitizes colorectal cancer to taxane treatment

Abstract Introduction: Colorectal cancer (CRC) is the third most common cancer diagnosed in both men and women. Dysregulation of the WNT/β-catenin pathway is the most commonly mutated pathway in CRC. R-spondins (RSPO), a family of secreted proteins, are enhancers of WNT signaling. Previous preclinical studies have demonstrated that WNT inhibition results in superior antitumor efficacy in combination with taxanes. This superior combinatorial activity with taxanes has been identified in lung, ovarian, breast and pancreatic cancers. Taxanes have been found to be generally ineffective in the clinical treatment of colorectal cancer. Analysis of the local microenvironment, or stem cell niche, has identified stromal cells as an abundant source of RSPO. In patient-derived tumor xenografts (PDX), the murine stromal cells replace human stromal cells and provide the microenvironment that supports tumor cell growth. We have classified PDX tumors as human RSPO3 high or low/negative using RNA sequencing and by expression studies. Inhibition of RSPO3 with OMP-131R10, a clinical stage therapeutic antibody which binds to human and murine RSPO3, has demonstrated efficacy in human RSPO3 high, APCWT PDX. Purpose: Common features in CRC are mutations in APC or β-catenin which are present in approximately 90% of cases. Tumors with RSPO3 genomic translocation and/or overexpression of RSPO3 are generally APC WT and β-cateninWT, and thus RSPO overexpression represents an alternative mechanism to upregulate the Wnt pathway. Alternatively, RSPO3 may be secreted by murine stromal cells, or by a rare population of tumor cells. In this study, we tested OMP-131R10 in combination with taxane treatment in human RSPO3 low CRC PDX models with APC or β-catenin mutations and in a PDX model with a RSPO3 translocation. Results: Anti-RSPO3 was highly effective in combination with nab-paclitaxel in a CRC model with an RSPO3 translocation and RSPO3 overexpression. Furthermore, the combination of OMP-131R10 and taxane treatment resulted in synergistic inhibition of tumor growth in 8/10 PDX models with APC or β -catenin mutations and low human tumor cell RSPO3 expression. Tumor regression or durable stable disease was evident in 6/8 models. Responsive models contained either heterozygous inactivating mutations in APC (6/8) or homozygous activating mutations in β-catenin (2/8). Combination of OMP-131R10 with paclitaxel potentiated mitotic arrest, enhanced terminal differentiation and reduced the tumorigenic cell frequency by 40 fold based on serial transplantation studies. Conclusion: Anti-RSPO3 (OMP-131R10) was active in the majority of CRC PDX models tested when combined with taxane treatment. These CRC models harbored mutations in APC and β-catenin. This data indicates that RSPO3 inhibition is an effective means to target the mutated Wnt pathway in CRC, and that antagonizing RSPO3 in combination with paclitaxel may improve survival for CRC patients. Citation Format: Marcus M. Fischer, V. Pete Yeung, Fiore Cattaruzza, Christopher Murriel, James W. Evans, Gilbert O'Young, Alayne Brunner, Min Wang, Belinda Cancilla, Ann Kapoun, Timothy Hoey. R-SPONDIN3 antagonism sensitizes colorectal cancer to taxane treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1911. doi:10.1158/1538-7445.AM2017-1911

Therapeutic Targeting of Tumor-Derived R-Spondin Attenuates β-Catenin Signaling and Tumorigenesis in Multiple Cancer Types

Deregulation of the β-catenin signaling has long been associated with cancer. Intracellular components of this pathway, including axin, APC, and β-catenin, are frequently mutated in a range of human tumors, but the contribution of specific extracellular ligands that promote cancer development through this signaling axis remains unclear. We conducted a reporter-based screen in a panel of human tumors to identify secreted factors that stimulate β-catenin signaling. Through this screen and further molecular characterization, we found that R-spondin (RSPO) proteins collaborate with Wnt proteins to activate β-catenin. RSPO family members were expressed in several human tumors representing multiple malignancies, including ovarian, pancreatic, colon, breast, and lung cancer. We generated specific monoclonal antibody antagonists of RSPO family members and found that anti-RSPO treatment markedly inhibited tumor growth in human patient-derived tumor xenograft models, either as single agents or in combination with chemotherapy. Furthermore, blocking RSPO signaling reduced the tumorigenicity of cancer cells based on serial transplantation studies. Moreover, gene-expression analyses revealed that anti-RSPO treatment in responsive tumors strongly inhibited β-catenin target genes known to be associated with cancer and normal stem cells. Collectively, our results suggest that the RSPO family is an important stimulator of β-catenin activity in many human tumors and highlight a new effective approach for therapeutically modulating this fundamental signaling axis.

Loss of TGF-β Signaling in Bone Marrow Mesenchymal Progenitors Promotes Adipocyte over Osteoblast Differentiation but Does Not Disrupt the HSC Niche

Hematopoietic stem cells (HSCs) reside in specialized microenvironments (niches) in the bone marrow. Several mesenchymal stromal cells have been implicated in hematopoietic niches, including osteoblasts, pericytes, CXCL12-abundant reticular (CAR) cells, and mesenchymal stem cells (MSCs). Members of the transforming growth factor (TGF) superfamily, in particular TGF-β, have a well-documented role in regulating osteoblast development. However, the contribution of TGF family member signaling to the establishment and maintenance of hematopoietic niches is largely unknown. Here, we characterize the role of transforming growth factor-β (TGF-β) signaling in mesenchymal stromal cells on the HSC niche.TGF-β receptor 2 (encoded by Tgfbr2) is required for all TGF-β signaling. To selectively disrupt TGF-β signaling in bone marrow mesenchymal stromal cells, we generated Osx-C re Tgfbr2fl/fl mice. Osx-Cre targets most bone marrow mesenchymal stromal cells (including osteoblasts, CAR cells, MSCs, pericytes, and adipocytes) but not endothelial cells or hematopoietic cells. Osx-C re Tgfbr2fl/fl mice are severely runted and most die by 4 weeks of age. We analyzed mice at 3 weeks, when the mice appeared healthy. Osteoblast number was severely reduced in Osx-C re Tgfbr2fl/fl mice, as assessed by histomorphometry and immunostaining for osteocalcin. Accordingly, microCT analysis demonstrated reduced tissue mineral density and cortical thickness of long bone and marked trabecularization of long bones in diaphyseal regions. Surprisingly, marrow adiposity, as measured by osmium tetroxide staining with microCT, was strikingly increased in Osx-C re Tgfbr2fl/fl mice. CAR cells are mesenchymal progenitors with osteogenic and adipogenic potential in vitro. To assess CAR cells, we generated Osx-Cre Tgfrb2fl/fl x Cxcl12gfp mice. Surprisingly, CAR cell number was significantly increased. However, despite the increase in CAR cells, the number of CFU-osteoblast (CFU-OB) in Osx-C re Tgfbr2fl/fl mice is nearly undetectable. Together, these data suggest that TGF-b signaling contributes to lineage commitment of mesenchymal progenitors. Specifically, our data suggest that TGF-β signaling suppresses commitment to the osteoblast lineage, while increasing adipogenic differentiation.We next asked whether alterations in bone marrow stromal cells present in Osx-C re Tgfbr2fl/fl mice affect HSC number or function. The increase in marrow adipocytes and loss of osteolineage cells is predicted to impair HSC maintenance, while the increase in CAR cells might augment HSCs. Osx-Cre Tgfrb2fl/fl mice have modest leukopenia, but normal red blood cell and platelet counts. Bone marrow and spleen cellularity are reduced, even after normalizing for body weight. The frequency of phenotypic HSCs (defined as Kit+ lineage- Sca+ CD34- Flk2- cells) is comparable to control mice. To assess HSC function, we performed competitive repopulation assays with bone marrow from Osx-Cre Tgfrb2fl/fl or control mice. Surprisingly, these data show that the long-term multi-lineage repopulating activity of HSCs from Osx-Cre Tgfrb2fl/fl mice is normal. Moreover, serial transplantation studies suggest that the self-renewal capacity of HSCs is normal. Thus, despite major alterations in mesenchymal stromal cell populations, the HSC niche is intact in Osx-Cre Tgfrb2fl/fl mice.Collectively, these data show that TGF-b signaling in mesenchymal progenitors is required for the proper development of multiple stromal cell populations that contribute to hematopoietic niches. Studies are underway to assess the impact of post-natal deletion of Tgfbr2 in mesenchymal stromal cell on hematopoietic niches. Since drugs that modulate the activity of TGF-b are in development, this research may suggest novel approaches to modulate hematopoietic niches for therapeutic benefit. DisclosuresNo relevant conflicts of interest to declare.

RNA Splicing Modulation Impairs Acute Myeloid Leukemia Stem Cell Maintenance

IntroductionDisease relapse is the leading cause of death in secondary AML (sAML), which evolves from antecedent hematologic disorders like myelodysplastic syndrome (MDS) or myeloproliferative neoplasms (MPNs) or following exposure to chemotherapy. Persistence of therapy-resistant leukemia stem cells (LSC) harboring enhanced survival and self-renewal capacity has been linked to high relapse rates in sAML. Previously, we showed that missplicing of a stem cell regulatory gene, GSK3 b, and splice isoform switching favoring pro-survival BCL2 family isoform expression promoted generation of therapy-resistant LSC (Abrahamsson et al PNAS 2009; Goff et al Cell Stem Cell 2013). However, whether aberrant pre-mRNA splicing promotes sAML LSC generation, in the absence of mutation, and if pharmacological splicing modulation impairs LSC maintenance, in a mutation-independent manner, has not been elucidated.Methods and ResultsComparative RNA-sequencing and gene set enrichment analyses revealed significant alterations in splicing factor gene expression in purified progenitors from untreated sAML compared with normal samples. In addition, using an isoform-specific alignment algorithm, we established a sAML LSC splice isoform expression signature that identified increased expression of select transcripts, e.g. CD82 and PTK2B. Thus, we investigated the LSC inhibitory efficacy of a stable, potent splicing modulatory agent, 17S -FD-895, in humanized AML LSC stromal co-culture and primagraft assays. Notably, there was a dose-dependent reduction in AML LSC (n=4) survival and self-renewal after in vitro 17S -FD-895 treatment, with a favorable therapeutic index compared to normal controls (n=3, p<0.01). Splicing reporter activity and PCR analyses revealed rapid and potent 17S -FD-895-induced alterations in splicing, promoting pro-apoptotic isoform expression and intron inclusion in the stem cell regulatory gene MCL1. Also, 17S -FD-895 restored normal expression patterns of PTK2B, and MCL1-L/S and BLC2-L/S expression ratios.Flow cytometric analyses in AML LSC primagraft models treated with 17S -FD-895 (5-10 mg/kg delivered intravenously in 3 doses over 2 weeks) revealed a decrease in human stem (CD45+ CD34+ CD38- Lin-, 68% reduction in the spleens of the 10 mg/kg group versus vehicle controls, n=5 mice per group, p<0.05) and progenitor (CD45+ CD34+ CD38+ Lin-, 80% reduction to nearly zero in the spleens of the 10 mg/kg group versus vehicle controls, p=0.08) cell frequencies. Furthermore, MCL1-L/S and BCL2-L/S expression ratios were significantly reduced in LSC-enriched fractions from 17S -FD-895-treated mice compared to vehicle controls. Consistent with a reduction in functional LSC burden after 17S -FD-895 treatment, subsequent serial transplantation studies showed a 47-65% reduction in leukemic burden in the hematopoietic tissues of recipients of CD34+ cells from mice in the 10 mg/kg treatment group versus vehicle controls (n=5 mice per group, p<0.05).ConclusionsHere we demonstrate that a potent and stable splicing modulatory agent, 17S -FD-895, normalized sAML-specific splice isoform expression patterns as well as MCL1-L/S and BLC2-L/S ratios. Moreover, pharmacologic splicing modulation reduced AML LSC survival and self-renewal in a dose-dependent manner in both in vitro and in vivo models with a favorable therapeutic index. Further evaluation of this compound as a splicing-targeted single agent or combined with standard of care therapy may reduce or eradicate LSC burden in therapy-resistant sAML. In addition, LSC-specific splice isoforms may represent important biomarkers that could be developed as companion diagnostics for splicing-targeted therapies in sAML and other recalcitrant malignancies. DisclosuresJamieson:Johnson & Johnson: Research Funding; GlaxoSmithKline: Research Funding.

Abstract C164: Dual targeting of the DLL4 and VEGF pathways with a bispecific monoclonal antibody inhibits tumor growth and reduces cancer stem cell frequency

Abstract Both Notch/Delta-like ligand 4 (DLL4) and vascular endothelial growth factor (VEGF) pathways play a critical role in angiogenesis and tumor growth. Due to differential regulatory effects of VEGF and DLL4 on the vasculature, blockade of DLL4 or VEGF signaling inhibits tumor growth by distinct mechanisms: anti-DLL4 treatment induces an abnormal increase of poorly perfused blood vessels, which results in nonproductive angiogenesis unable to support tumor growth, whereas anti-VEGF therapy significantly decreases vasculature reducing the blood supply to tumors. In addition, DLL4-Notch signaling plays a key role in the maintenance of cancer stem cells. We have recently developed a bispecific monoclonal antibody that targets both human DLL4 and human VEGF (OMP-305B83). In vitro, this antibody exhibited low nanomolar binding affinity to hVEGF and hDLL4, and reduced human endothelial cell proliferation induced by VEGF. The bispecific antibody demonstrated significant in vivo anti-tumor efficacy in various solid tumors, induced tumor regression, decreased the frequency of tumor initiating cells, and delayed tumor recurrence following termination of chemotherapy. Analysis of tumor vasculature after treatment with anti-DLL4/VEGF revealed inhibition of vascular gene expression and endothelial cell proliferation, indicating that the anti-VEGF effect on the vasculature is dominant over the anti-DLL4 effect. Notably, at doses where both anti-DLL4 and anti-VEGF alone produces suboptimal anti-tumor effect, dual targeting resulted in additive tumor growth inhibition. The combination of anti-DLL4 and anti-VEGF resulted in broad spectrum efficacy in many different solid tumor types including breast, colon, ovarian and pancreatic tumors. Notably, serial transplantation studies indicated that the anti-cancer stem cell activity of anti-DLL4 was retained with the bispecific. In safety studies, OMP-305B83 demonstrated an improved cardiac profile in cynomolgus monkeys compared to anti-DLL4 with reduction of endothelial hyperplasia and suppression of vascular-related gene upregulation in the heart. These results indicate that our bispecific anti-DLL4/VEGF is broadly efficacious and may be useful for treatment of a variety of tumor types. We are currently enrolling patients with advanced refractory solid tumors in a Phase 1a clinical trial. Citation Format: Wan-Ching Yen, Marcus M. Fischer, Gretchen Argast, Breanna Wallace, Min Wang, Rene Meisner, John Lewicki, Ann M. Kapoun, Austin Gurney, Timothy Hoey. Dual targeting of the DLL4 and VEGF pathways with a bispecific monoclonal antibody inhibits tumor growth and reduces cancer stem cell frequency. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C164.

Abstract 2807: Oral silibinin inhibits tumorigenic potential of colon cancer stem cells

Abstract Recent studies by our group have demonstrated that silibinin, a non-toxic, chemopreventive agent, has an inhibitory effect on the self-renewal capacity and differentiation potential of the cancer stem cell (CSC) population of CRC cell lines. However, these observations were under in vitro conditions. To overcome the limitations of these findings, in the present study, we assessed oral silibinin effect on tumorigenic potential of colon CSC. For this, we employed phenotypic cell isolation strategy to separate CD44+EpCAMhigh (stem cells enriched) HT-29 CRC cells by flow cytometry/magnetic activated sorting. These sorted and unsorted cells were used to generate ectopic xenograft tumors in NOD/SCID mice and silibinin effect on initiation, growth, and metabolism of these tumors was assessed. Briefly, unsorted and sorted population (CD44+EpCAMhigh) from CRC HT29 cells purified using FACS as per defined protocols were injected sub-cutaneously into NOD/SCID mice (6-8 weeks old), and animals were orally fed with vehicle or silibinin (200 mg/ kg body weight), starting 24 h post-injection (n = 14 per group). Unsorted cells were similarly injected as 2 x106 cells/ mouse. Once xenograft started growing, tumor sizes and volumes were calculated. Tumor latency (time from injection to detection of palpable tumor) were also noted. In general, experiment was terminated by 40 days post cell injections or terminated earlier if tumors reach a maximum diameter of 15 mm. To support tumor latency and tumor size results obtained visibly, anatomical proton-density magnetic resonance imaging (MRI) scans (day 2, 7 and 14) were acquired. Functional imaging (performed for 60-80 mm3 tumor and by end of study) was performed to report on tumor cellularity by diffusion-weighted (DW-), vascularity by dynamic contrast-enhanced (DCE-) MRI and glycolytic activity by 18Fluorine-2-Deoxy-Glucose (FDG) positron emission tomography (PET). Tumors harvested at the end of above study were subjected to live cell sorting analysis to determine the percentage of CSC population (CD44+EpCAMhigh cells) in xenografts. To determine oral silibinin effect on CSC self-renewal in serial transplantation studies, these CSC enriched fractions and the unsorted population of tumor cells were transplanted back into NOD/SCID mice and followed for initiation, growth, and tumor phenotype with or without silibinin treatment. Sorted CSC and unsorted tumor cells from both control and silibinin-treated mice were also subjected to in vitro experiments to determine their self-renewal and differentiation capacity. Results show that indeed silibinin feeding has an inhibitory effect on tumorigenic potential of colon CSC; a decrease in latency as well as size/ volume of tumor was observed. This effect was associated with a reduced number of CSC's in silibinin-fed tumors, a decrease in tumor metabolic activity and an inhibition of angiogenic feed to the tumors. Citation Format: Sushil Kumar, Dileep Kumar, Komal Raina, Natalie J. Serkova, Chapla Agarwal, Rajesh Agarwal. Oral silibinin inhibits tumorigenic potential of colon cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2807. doi:10.1158/1538-7445.AM2015-2807

Comparison of Different Cytokine Conditions Reveals Resveratrol as a New Molecule for Ex Vivo Cultivation of Cord Blood-Derived Hematopoietic Stem Cells

Human cord blood (CB)-derived hematopoietic stem cells (HSCs) are an interesting source for HSC transplantation. However, the number of collected CB-HSCs is often too low for one transplantation; therefore, ex vivo expansion of CB-HSCs is desirable. Current expansion protocols are based on the use of cytokine combinations, including insulin-like growth factor-binding protein 2 (IGFBP2) and angiopoietin-like proteins, or combinations with "small molecules" such as stemregenin-1. The aim of our project was to compare the potential of different CB-HSC expansion strategies side-by-side by phenotypical analysis in vitro and serial engraftment properties in NOD/SCID/IL2rg-/- (NSG) immunodeficient mice. We further identified resveratrol, a naturally occurring polyphenol, as a new, alternative small molecule combined with cytokines to facilitate serum-free ex vivo expansion of human CB-HSCs. The cultivation in resveratrol preserved the CB-HSC phenotype in vitro most efficiently and was ∼2 times more potent than commonly used cytokine conditions (including stem cell factor, thrombopoietin, Fms-related tyrosine kinase 3 ligand, interleukin-6) and the recently established serum-free culture, including IGFBP2 and angiopoietin-like 5. Serial transplantation studies further confirmed resveratrol to support robust multilineage engraftment in primary and secondary NSG recipients. Therefore, our work proposes resveratrol as a new small molecule for improved ex vivo culture and modification of human HSCs based on an efficient ex vivo propagation of the HSC fate. Human cord blood (CB)-derived hematopoietic stem cells (HSCs) are an important source for HSC transplantations but restricted in their usage because of their low numbers. In gene therapy, modifications of HSCs relies on their ex vivo modification without losing their stemness properties. Therefore, ex vivo cultivation and expansion of CB-HSCs is important for their effective application in HSC transplantation and gene therapy. Several promising protocols for serum-free cultivation of HSCs using different combinations of cytokines or so-called small molecules are described. A direct comparison was performed of three described serum-free cytokine conditions, demonstrating that the natural occurring polyphenol resveratrol is able to support ex vivo cultivation of CB-HSCs. The results show that resveratrol is an additional candidate for improving ex vivo cultures of HSCs for transplantation and gene therapeutic applications in the future.

A Shot in the Bone Corrects a Genetic Disease

A Shot in the Bone Corrects a Genetic Disease

CD200 May be a Potential Target for Therapy in Standard Risk Childhood ALL

Multiagent therapies for children with acute lymphoblastic leukaemia (ALL) have resulted in impressive remission rates of around 90%. Unfortunately, relapse rates remain high and most children who relapse will not survive. Innovative antibody and chimeric antigen receptor (CAR) modified T-cell therapies have shown promising results in ALL. However, this is a heterogeneous disease and several groups have shown ALL evolves in a branching fashion. Moreover, some leukaemia stem cell (LSC) populations do not express antigens that are the targets of developing therapies such as CD19, CD20, and CD22. Consequently, these cells may be largely unaffected and could provide a reservoir of resistant cells, from which relapse may develop. The diversity of reported LSC populations may be a reason for treatment failure and improvements in therapy will require targeting of all cells that have the potential to initiate and maintain this disease. In an attempt to identify more appropriate therapeutic targets we conducted an extensive microarray screen of several LSC subpopulations and bulk leukaemia cells from a group of paediatric patients with favourable cytogenetic abnormalities. This screen identified a number of genes that were up-regulated in all LSC subpopulations compared to levels in normal haemopoietic cells. The up-regulated genes encode for cell surface antigens, including the type-1 membrane protein, CD200 and signalling pathways. Expression of CD200 was 12-fold higher in bulk ALL cells compared to normal BM cells (p=0.001). CD200 is a key immunosuppressive molecule that has been implicated as a poor prognostic factor in multiple myeloma and acute myeloid leukaemia. To validate the microarray results, we assessed expression of CD200 in 20 BCP-ALL cases (11 pre B, 9 c-ALL) by flow cytometry and in functional assays. Fifteen of these cases were classified as MRD low risk and had favourable cytogenetic abnormalities and 5 were classified as high risk by MRD and cytogenetic abnormalities. ALL cells were stained with antibodies against CD34, CD19 and CD200 and cells were sorted on the basis of expression/lack of expression of CD34/CD200. The functional ability of the sorted populations was assessed in NSG mice and serial transplantation experiments were performed to assess self-renewal capacity. Overall, the flow cytometric analyses confirmed the microarray data in that expression of CD200 was high in this patient cohort (60±6.6%) compared to normal BM (0.1%±0.04, p ≤ 0.001), regardless of cytogenetic abnormality or MRD risk status. In standard risk cases, BM engraftment was achieved with the CD34+/CD200+ (2-66% human leukaemia) and CD34-/CD200+ (10-26%) subpopulations only, using inocula of 4x104-107 cells. No engraftment was attained using similar numbers of CD200- cells, regardless of expression of CD34 or CD19 in this group. Interestingly, when cells from the 5 patients defined as high risk were inoculated, engraftment was achieved using all four sorted subpopulations. The levels of leukaemia engraftment in these cases ranged from 2-95% using 1x103-5x106 cells and levels were similar across all subpopulations in individual patient samples. Results from serial transplantation studies to date demonstrate the sorted subpopulations were capable of self-renewal. As with the primary transplants, all four subpopulations from high risk samples engrafted while this ability was restricted to CD200+ subpopulations in standard risk cases. FISH analyses have confirmed all the engrafting cells had an abnormal karyotype. These data provide further evidence that surface antigen immunophenotype is not a good predictor of function in paediatric ALL, particularly in high risk cases. However, in standard risk cases it may be possible to eliminate LSC by targeting CD200. Treating human chronic lymphocytic leukaemia cells with anti-CD200 antibodies has been shown to prevent engraftment in NSG mice. Our data suggests that investigation of the effects of anti-CD200 in standard risk childhood ALL are warranted. DisclosuresNo relevant conflicts of interest to declare.

Selenium suppresses leukemia through the action of endogenous eicosanoids.

Eradicating cancer stem-like cells (CSC) may be essential to fully eradicate cancer. Metabolic changes in CSC could hold a key to their targeting. Here, we report that the dietary micronutrient selenium can trigger apoptosis of CSC derived from chronic or acute myelogenous leukemias when administered at supraphysiologic but nontoxic doses. In leukemia CSC, selenium treatment activated ATM-p53-dependent apoptosis accompanied by increased intracellular levels of reactive oxygen species. Importantly, the same treatment did not trigger apoptosis in hematopoietic stem cells. Serial transplantation studies with BCR-ABL-expressing CSC revealed that the selenium status in mice was a key determinant of CSC survival. Selenium action relied upon the endogenous production of the cyclooxygenase-derived prostaglandins Δ(12)-PGJ2 and 15d-PGJ2. Accordingly, nonsteroidal anti-inflammatory drugs and NADPH oxidase inhibitors abrogated the ability of selenium to trigger apoptosis in leukemia CSC. Our results reveal how selenium-dependent modulation of arachidonic acid metabolism can be directed to trigger apoptosis of primary human and murine CSC in leukemia.

Abstract 247: ADAR1 promotes malignant progenitor reprogramming in chronic myeloid leukemia.

Abstract The molecular etiology of human progenitor reprogramming into self-renewing leukemia stem cells (LSC) has remained elusive. While DNA sequencing has uncovered spliceosome gene mutations that promote alternative splicing and portend leukemic transformation, isoform diversity may also be generated by RNA editing mediated by adenosine deaminase acting on RNA (ADAR) enzymes that regulate stem cell maintenance. In this study, whole transcriptome sequencing of normal, chronic phase (CP) and serially transplantable blast crisis (BC) chronic myeloid leukemia (CML) progenitors revealed increased interferon-γ pathway gene expression in concert with BCR-ABL amplification, enhanced expression of the interferon responsive ADAR1 p150 isoform and a propensity for increased A-to-I RNA editing during CML progression. Lentiviral overexpression experiments demonstrate that ADAR1 p150 promoted expression of the myeloid transcription factor PU.1 and induced malignant reprogramming of myeloid progenitors. Moreover, enforced ADAR1 p150 expression was associated with production of a mis-spliced form of GSK3β implicated in LSC self-renewal. Finally, functional serial transplantation and shRNA studies demonstrate that ADAR1 knockdown impaired in vivo self-renewal capacity of BC CML progenitors. Together these data provide a compelling rationale for developing ADAR1-based LSC detection and eradication strategies. Citation Format: Qingfei Jiang, Leslie A. Crews, Christian L. Barrett, Angela Court-Recart, Daniel Goff, Anil Sadarangani, Jessica Rusert, Sheldon Morris, Lawrence Goldstein, Hye-Jung Chun, Marco Marra, Kelly Fraser, Kim-Hien Dao, Mark Minden, Catriona Jamieson. ADAR1 promotes malignant progenitor reprogramming in chronic myeloid leukemia. [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 247. doi:10.1158/1538-7445.AM2013-247

Abstract 907: Efficacy of SAR302503, a JAK2 inhibitor, in primary human acute myeloid leukemia xenografts.

Abstract Small molecule inhibitors of Janus kinase 2 (JAK2) such as Ruxolitinib and SAR302503 have demonstrated efficacy in clinical trials for treatment of myeloproliferative disorders. Activated JAK2 signaling has been reported in some acute myeloid leukemia (AML) samples even though JAK2 mutations are relatively rare in AML. Whether JAK2 inhibitors are effective in AML, particularly against the disease-sustaining leukemia stem cells (LSC), is not clear. We report that SAR302503 (Sanofi, Cambridge MA), an orally administered small molecule JAK2 inhibitor, shows efficacy in a xenograft model of human AML established by intrafemoral injection of primary AML cells into sublethally irradiated NOD.SCID mice. The AML samples tested were of multiple subtypes with heterogeneous cytogenetic and molecular abnormalities. Starting 2 weeks post transplantation to permit establishment of an AML graft, mice received twice daily oral gavage with 60 mg/kg SAR302503 or vehicle alone (0.5% methylcellulose) for 14 consecutive days. In 17 of 34 AML samples, SAR302503 treatment reduced leukemic engraftment in the injected femur (56-94% relative reduction, RR, compared to controls; p&amp;lt;0.05) as well as non-injected bones (30-95% RR; p&amp;lt;0.05). 5 additional samples exhibited a partial response (&amp;lt;50% RR in injected femur and 31-64% RR in non-injected bones, p&amp;lt;0.05). In preliminary serial transplantation studies, AML cells harvested from SAR302503-treated primary mice showed reduced ability to generate a leukemic graft in untreated secondary mice compared to controls, suggesting that JAK2 inhibition reduces LSC function and/or survival. Given the heterogeneous response to SAR302503 treatment observed in vivo, we carried out phosphoflow cytometric analysis of patient samples to identify biomarkers that could predict drug response. AML samples that were sensitive to JAK2 inhibition in xenotransplantation assays exhibited high basal levels of pSTAT5, often in only a small subset of cells, that were rapidly decreased by SAR302503 treatment in vitro, whereas non-responding samples showed low levels of pSTAT5, suggesting that pSTAT5 is a useful drug response biomarker. Our results demonstrate the potential of SAR302503 to target AML cells including LSCs in a broad cross section of AML patients, and warrant further studies to identify responders and non-responders and better characterize proteomic biomarkers of drug response. The approach we have taken, which focuses on large-scale analysis of primary samples using state-of-the-art xenograft assays, offers a new paradigm for preclinical drug development to identify both novel agents that effectively target LSCs and the patients most likely to benefit from targeted treatment. Citation Format: Weihsu C. Chen, Andreea C. Popescu, Yan Xing, Gitte Gerhard, Julie Yuan, Mark Minden, Cynthia Guidos, Donna E. Hogge, John E. Dick, Jean CY Wang. Efficacy of SAR302503, a JAK2 inhibitor, in primary human acute myeloid leukemia xenografts. [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 907. doi:10.1158/1538-7445.AM2013-907

Abstract 3725: Anti-DLL4 (demcizumab) inhibits tumor growth and reduces cancer stem cell frequency in patient-derived ovarian cancer xenografts.

Abstract Ovarian cancer in a major cause cancer related death in women and this disease is often characterized by resistance to chemotherapy and tumor recurrence. Cancer stem cells (CSCs) have been shown to be relatively insensitive to chemotherapeutic agents. Several lines of evidence indicate that aberrant Notch signaling plays a major role in ovarian cancer progression and resistance to standard therapies. DLL4 is a key ligand that activates the Notch pathway and has been shown to regulate both CSC function and tumor angiogenesis. DLL4 expression in tumor and endothelial cells has also been associated with resistance to bevacizumab treatment. In these studies, we investigated the role of anti-DLL4 in a panel of patient-derived ovarian cancer xenograft models. We utilized anti-human DLL4 (OMP-21M18) and anti-murine DLL4 to block Notch signaling in both the tumor and stromal/vascular cells in the xenografts. We found that anti-DLL4 treatment was broadly efficacious in these ovarian cancer models, significantly inhibiting tumor growth both as a single agent and in combination with paclitaxel. We carried out serial transplantation studies to investigate the effect on cancer stem cells and found that anti-DLL4 in combination with paclitaxel profoundly reduced the tumor initiating cell frequency. In contrast, treatment with paclitaxel alone had the opposite effect and increased ovarian CSC frequency. These data suggest that anti-DLL4 treatment sensitizes chemoresistant ovarian tumorigenic cells to therapy and provide compelling evidence that anti-DLL4 (demcizumab) is a promising strategy for the clinical treatment of ovarian cancer. Citation Format: Wan-Ching Yen, Marcus M. Fischer, Jalpa Shah, Jie Wei, Jennifer Cain, Pete Yeung, Lucia Beviglia, Belinda Cancilla, Ann Kapoun, John Lewicki, Austin Gurney, Timothy Hoey. Anti-DLL4 (demcizumab) inhibits tumor growth and reduces cancer stem cell frequency in patient-derived ovarian cancer xenografts. [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 3725. doi:10.1158/1538-7445.AM2013-3725

ADAR1 promotes malignant progenitor reprogramming in chronic myeloid leukemia

The molecular etiology of human progenitor reprogramming into self-renewing leukemia stem cells (LSC) has remained elusive. Although DNA sequencing has uncovered spliceosome gene mutations that promote alternative splicing and portend leukemic transformation, isoform diversity also may be generated by RNA editing mediated by adenosine deaminase acting on RNA (ADAR) enzymes that regulate stem cell maintenance. In this study, whole-transcriptome sequencing of normal, chronic phase, and serially transplantable blast crisis chronic myeloid leukemia (CML) progenitors revealed increased IFN-γ pathway gene expression in concert with BCR-ABL amplification, enhanced expression of the IFN-responsive ADAR1 p150 isoform, and a propensity for increased adenosine-to-inosine RNA editing during CML progression. Lentiviral overexpression experiments demonstrate that ADAR1 p150 promotes expression of the myeloid transcription factor PU.1 and induces malignant reprogramming of myeloid progenitors. Moreover, enforced ADAR1 p150 expression was associated with production of a misspliced form of GSK3β implicated in LSC self-renewal. Finally, functional serial transplantation and shRNA studies demonstrate that ADAR1 knockdown impaired in vivo self-renewal capacity of blast crisis CML progenitors. Together these data provide a compelling rationale for developing ADAR1-based LSC detection and eradication strategies.