Human Bone Marrow Progenitor Cells Research Articles

Decoding human bone marrow hematopoietic stem and progenitor cells from fetal to birth

Bone marrow (BM) is the dominant site of hematopoiesis after 20 post-conception weeks (PCWs), but the intricacies of hematopoietic development in fetal BM up to birth and its involvement in malignancies remain unknown. Here, we compared the single-cell transcriptomic profile of BM hematopoietic stem and progenitor cells (HSPCs) at the early (12-14 PCW), middle (19-22 PCW) second trimester, and the neonatal stage. The stemness of hematopoietic stem cell and multipotent progenitor (HSC/MPP) is established at the middle second trimester, then maintained until birth. Furthermore, differentiation potentials toward three lineages are enhanced after the middle second trimester for birth, accompanied by the upregulation of aerobic metabolism. Notably, decreased stemness in HSCs/MPPs and higher interferon signals in progenitors at the early second trimester rendered the HSPCs more proximal to leukemogenesis. Collectively, our work elucidated the dynamics of fetal hematopoiesis in preparation for birth, offering valuable insights into the pathological processes underlying leukemia.

Abstract 4534: HSK40495, a highly selective PARP1 inhibitor with improved hematopoietic safety for the treatment of HRD cancers

Abstract It is well known that several PARP inhibitors have been approved for tumors with homologous recombination repair deficiencies (HRD), such as BRCA mutations. However, the common side effect of these inhibitors is hematologic toxicity, which limits their therapeutic index and combination potential with first-line chemotherapies. One possible reason is that currently approved PARP inhibitors do not specifically target PARP1, and might also affect other PARP family members. Recent studies have shown that the synthetic lethality caused by PARP trapping with HRD is mainly dependent on PARP1 inhibition, but not other PARP family members. Accordingly, we developed a selective PARP1 inhibitor that effectively inhibits tumor growth with less hematologic toxicity. HSK40495 is a potent PARP1 inhibitor with highly selectivity of PARP2. HSK40495 inhibited PARP1 enzymatic activity with a single digit nanomolar potency and with greater than 5000-fold selectivity against PARP2 in the protein binding assay. In addition, HSK40495 exhibited potent anti-proliferation activity in both DLD1(BRCA KO) cell and MDA-MB-436 cell, while had minimal effects against BRCA-WT DLD1 cell up to 30 µM in an anti-proliferation assay. In a subcutaneous MDA-MB-436 xenograft tumor model, HSK40495 administered orally once daily achieved excellent tumor growth inhibition of 100% at 3mg/kg. HSK40495 demonstrated significant PARylation inhibition in tumor tissues. Importantly, in CD34+ human bone marrow progenitor cell, HSK40495 showed improved cytotoxicity when compare to first-generation PARP inhibitor, Talazoparib. Meanwhile, in a lineage-specific hematotoxicity assay, HSK40495 exhibited improved cytotoxic safety in the process of differentiation of myeloid, erythroid and megakaryocyte compared with Talazoparib treatment. In summary, HSK40495, a highly selective PARP1inhibitor over other PARP family members, exhibits excellent pharmacological and safety properties, and also has the favorable pharmacokinetics profile in preclinical studies. All these findings suggest that HSK40495 has the great potential to be a promising PARP1 inhibitor for further development in the patients with HRD tumors. Citation Format: Fan Li, Xiaoli Gou, Lei Chen, Qingyuan Meng, Yao Li, Hongjiao Dong, Pingming Tang, Ju Wang, Pangke Yan. HSK40495, a highly selective PARP1 inhibitor with improved hematopoietic safety for the treatment of HRD cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4534.

Injectable biomaterial induces regeneration of the intervertebral disc in a caprine loaded disc culture model.

Back pain is the leading cause of disability with half of cases attributed to intervertebral disc (IVD) degeneration, yet currently no therapies target this cause. We previously reported an ex vivo caprine loaded disc culture system (LDCS) that accurately represents the cellular phenotype and biomechanical environment of human IVD degeneration. Here, the efficacy of an injectable hydrogel system (LAPONITE® crosslinked pNIPAM-co-DMAc, (NPgel)) to halt or reverse the catabolic processes of IVD degeneration was investigated within the LDCS. Following enzymatic induction of degeneration using 1 mg mL-1 collagenase and 2 U mL-1 chondroitinase ABC within the LDCS for 7 days, IVDs were injected with NPgel alone or with encapsulated human bone marrow progenitor cells (BMPCs). Un-injected caprine discs served as degenerate controls. IVDs were cultured for a further 21 days within the LDCS. Tissues were then processed for histology and immunohistochemistry. No extrusion of NPgel was observed during culture. A significant decrease in histological grade of degeneration was seen in both IVDs injected with NPgel alone and NPgel seeded with BMPCs, compared to un-injected controls. Fissures within degenerate tissue were filled by NPgel and there was evidence of native cell migration into injected NPgel. The expression of healthy NP matrix markers (collagen type II and aggrecan) was increased, whereas the expression of catabolic proteins (MMP3, ADAMTS4, IL-1β and IL-8) was decreased in NPgel (±BMPCs) injected discs, compared to degenerate controls. This demonstrates that NPgel promotes new matrix production at the same time as halting the degenerative cascade within a physiologically relevant testing platform. This highlights the potential of NPgel as a future therapy for IVD degeneration.

Continuous treatment with abemaciclib leads to sustained and efficient inhibition of breast cancer cell proliferation.

Abemaciclib is an oral, selective cyclin-dependent kinase 4 & 6 inhibitor (CDK4 & 6i), approved for hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2–) advanced breast cancer (ABC) as monotherapy for endocrine refractory disease, and with endocrine therapy (ET) for initial treatment and after progression on ET. Abemaciclib has also shown clinical activity in combination with ET in patients with high risk early BC (EBC). Here, we examined the preclinical attributes of abemaciclib and other CDK4 & 6i using biochemical and cell-based assays. In vitro, abemaciclib preferentially inhibited CDK4 kinase activity versus CDK6, resulting in inhibition of cell proliferation in a panel of BC cell lines with higher average potency than palbociclib or ribociclib. Abemaciclib showed activity regardless of HER2 amplification and phosphatidylinositol 3-kinase (PI3KCA) gene mutation status. In human bone marrow progenitor cells, abemaciclib showed lower impact on myeloid maturation than other CDK4 & 6i when tested at unbound concentrations similar to those observed in clinical trials. Continuous abemaciclib treatment provided profound inhibition of cell proliferation, and triggered senescence and apoptosis. These preclinical results support the unique efficacy and safety profile of abemaciclib observed in clinical trials.

Discovery of 5-{4-[(7-Ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl]piperazin-1-yl}-N-methylpyridine-2-carboxamide (AZD5305): A PARP1-DNA Trapper with High Selectivity for PARP1 over PARP2 and Other PARPs.

Poly-ADP-ribose-polymerase (PARP) inhibitors have achieved regulatory approval in oncology for homologous recombination repair deficient tumors including BRCA mutation. However, some have failed in combination with first-line chemotherapies, usually due to overlapping hematological toxicities. Currently approved PARP inhibitors lack selectivity for PARP1 over PARP2 and some other 16 PARP family members, and we hypothesized that this could contribute to toxicity. Recent literature has demonstrated that PARP1 inhibition and PARP1-DNA trapping are key for driving efficacy in a BRCA mutant background. Herein, we describe the structure- and property-based design of 25 (AZD5305), a potent and selective PARP1 inhibitor and PARP1-DNA trapper with excellent in vivo efficacy in a BRCA mutant HBCx-17 PDX model. Compound 25 is highly selective for PARP1 over other PARP family members, with good secondary pharmacology and physicochemical properties and excellent pharmacokinetics in preclinical species, with reduced effects on human bone marrow progenitor cells in vitro.

Abemaciclib, a CDK4 and 6 inhibitor with unique pharmacological properties for breast cancer therapy.

e12506 Background: Breast cancer is the second most common cancer worldwide. Pharmacologically targeting cyclin-dependent kinases 4 and 6 (CDK4 & 6) has proven to be a successful therapeutic approach in patients with estrogen receptor positive (ER+) breast cancer. Differences in both efficacy and toxicity among the available CDK4 & 6 inhibitors has generated interest in a biological explanation. Abemaciclib is an adenosine triphosphate-competitive, reversible, selective inhibitor of CDK4 & 6 that has shown antitumor activity as a single agent and in combination with standard endocrine therapy (ET), in hormone receptor positive (HR+) metastatic breast cancer patients including those with ET resistance, and in combination with ET in high-risk early breast cancer patients. This study examines attributes of abemaciclib and other CDK4 & 6 inhibitors. Methods: The potency of abemaciclib for CDK4 was evaluated using biochemicals and breast cancer cell-based assays. Additionally, different combinations with an anti-estrogen therapy (e.g., tamoxifen, fulvestrant) were analyzed in an in vitro palbociclib (CDK4 & 6 inhibitor)-resistant breast cancer cell model, as well as in a set of CDK4 & 6 sensitive breast cancer cell models. Using cell-free assays, high content imaging and flow cytometry approaches, a subset of markers were monitored to characterize the phenotype of sensitive cell lines in a continuous dose schedule. Results: In in vitro, cell-free assays, abemaciclib shows selectivity for CDK4 over CDK6, and in cell-based assays, abemaciclib preferentially inhibits the proliferation of cells dependent on the presence of CDK4, not CDK6. Abemaciclib inhibits cell proliferation in a wide range of breast cancer cell lines, showing activity regardless of human epidermal growth factor receptor 2 (HER2) and PI3KCA gene mutation status. Furthermore, in a cell line resistant to palbociclib, abemaciclib in combination with fulvestrant (ET) restores CDK4 & 6 sensitivity, leading to cell senescence and cell death. Finally, in human bone marrow progenitor cells, abemaciclib shows a lesser impact on myeloid maturation than other CDK4 & 6 inhibitors, palbociclib and ribociclib, allowing for continuous dosing. Conclusions: In pre-clinical experiments, abemaciclib is a potent cell growth inhibitor, inhibiting preferentially the CDK4/CyclinD1 complex, leading to cell senescence and cell death. These pre-clinical results support the differentiated safety and efficacy profile of abemaciclib observed in clinical trials.

The hematopoietic stem cell marker VNN2 is associated with chemoresistance in pediatric B-cell precursor ALL

AbstractMost relapses of acute lymphoblastic leukemia (ALL) occur in patients with a medium risk (MR) for relapse on the Associazione Italiana di Ematologia e Oncologia Pediatrica and Berlin-Frankfurt-Münster (AIEOP-BFM) ALL protocol, based on persistence of minimal residual disease (MRD). New insights into biological features that are associated with MRD are needed. Here, we identify the glycosylphosphatidylinositol-anchored cell surface protein vanin-2 (VNN2; GPI-80) by charting the cell surface proteome of MRD very high-risk (HR) B-cell precursor (BCP) ALL using a chemoproteomics strategy. The correlation between VNN2 transcript and surface protein expression enabled a retrospective analysis (ALL-BFM 2000; N = 770 cases) using quantitative polymerase chain reaction to confirm the association of VNN2 with MRD and independent prediction of worse outcome. Using flow cytometry, we detected VNN2 expression in 2 waves, in human adult bone marrow stem and progenitor cells and in the mature myeloid compartment, in line with proposed roles for fetal hematopoietic stem cells and inflammation. Prospective validation by flow cytometry in the ongoing clinical trial (AIEOP-BFM 2009) identified 10% (103/1069) of VNN2+ BCP ALL patients at first diagnosis, primarily in the MRD MR (48/103, 47%) and HR (37/103, 36%) groups, across various cytogenetic subtypes. We also detected frequent mutations in epigenetic regulators in VNN2+ ALLs, including histone H3 methyltransferases MLL2, SETD2, and EZH2 and demethylase KDM6A. Inactivation of the VNN2 gene did not impair leukemia repopulation capacity in xenografts. Taken together, VNN2 marks a cellular state of increased resistance to chemotherapy that warrants further investigations. Therefore, this marker should be included in diagnostic flow cytometry panels.

Nonclinical safety assessment of AMG 553, an investigational anti-FLT3 CAR-T therapy.

7032 Background: FMS-like tyrosine kinase 3 (FLT3) is a tyrosine-protein kinase involved in hematopoiesis. In acute myeloid leukemia (AML), higher FLT3 transcript levels, independent of the presence of FLT3 mutations, correlate with higher leukocyte counts and higher degrees of bone marrow infiltration by leukemic cells. FLT3 protein is detectable on the cell surface of more than 80% of leukemia isolates from adult AML patients whereas FLT3 mutations are present only in approximately one third of patients. AMG 553 is a novel, investigational, adoptive cellular immunotherapy for the treatment of relapsed refractory AML, consisting of autologous T cells genetically modified ex vivo to express a transmembrane chimeric antigen receptor (CAR) to target FLT3 protein on the surface of AML cells irrespective of FLT3 mutational status. Methods: The nonclinical safety evaluation of AMG 553 includes assessment of FLT3 expression in normal tissues; in vitro assessment of cytotoxicity against human cells with or without FLT3 expression; toxicology studies in cynomolgus monkeys administered autologous T-cells transduced with an anti-FLT3 CAR or administered anti-FLT3 bi-specific T-cell engager (BiTE) antibodies. Results: Assessment of FLT3 expression in normal tissues indicates that FLT3 protein is detected on the cell surface of a subpopulation of human bone marrow hematopoietic stem and progenitor cells; expression in other tissues is cytoplasmic and not anticipated to be accessible to AMG 553. AMG 553-induced cytotoxicity was only observed in a percentage of primary bone marrow CD34+ cells. There was no evidence of CAR-T cell-mediated toxicity, expansion, or persistence in cynomolgus monkeys likely due to restricted cell surface FLT3 protein expression in healthy animals which demonstrates the limited value of such studies for this target. All findings in toxicology studies with anti-FLT3 BiTEs were consistent with the targeted killing of cells expressing FLT3 on the plasma membrane. Conclusions: The nonclinical safety data support AMG 553 as a novel therapeutic to target FLT3 protein on AML cells irrespective of FLT3 mutational status, while only affecting a percentage of normal hematopoietic stem and progenitor cells.

MMP9 Inhibition Rescues the Erythroid Defect in RPS14-Deficient Del(5q) MDS Models

The del(5q) myelodysplastic syndrome (MDS) is a unique subtype of MDS characterized by an interstitial deletion of the long arm of chromosome 5 with macrocytic anemia, normal to increased platelet count, and hypoloblated megakaryocytes. Although haploinsufficiency of several genes encoded within the commonly deleted region have been implicated in the hematologic phenotype of del (5q) MDS, the deletion of the RPS14 gene, which encodes a component of the 40S ribosomal subunit, is a key determinant of the severe macrocytic anemia seen in patients. Lenalidomide is FDA-approved and highly effective for the treatment for del(5q) MDS. However, there are rare resistant cases resulting from p53 mutations in del(5q) MDS patients and 50% of patients acquire resistance within 2 to 3 years. Given these limitations of lenalidomide treatment, as well as frequent side effects, we sought to identify novel targets for the treatment of del(5q) MDS patients by performing an in vivo drug screening assay using a zebrafish model of MDS.We generated stable genetic mutation of zebrafish RPS14 using CRISPR-Cas9 gene targeting technique. This zebrafish model mirrors the anemic phenotype seen in the 5q-syndrome. We found that mmp9 gene expression was abnormally induced by RPS14 deficiency in zebrafish and MMP9 inhibitors partially rescued erythroid defects in RPS14-deficient zebrafish. To study the role of MMP in human hematopoietic stem cells, we treated MMP9 inhibitors in RPS14 or luciferase control knockdown human bone marrow stem and progenitor (BMCD34+) cells for 6 days and then evaluated erythroid development. Our results showed that treatment with MMP9 inhibitors in RPS14 knockdown CD34+ cells significantly increased erythroid colony numbers compared with DMSO control (1.5 to 2.5-fold increase). Similarly, FACS analysis demonstrated increased the CD71+ erythroid population in RPS14 downregulated cells by MMP9 inhibitor treatment (4-fold increase compared to control), but no difference in the CD11b+ myeloid population. This data suggests that MMP9 inhibitors have specific roles in the erythroid development in RPS14 deficient cells.To understand the mechanism of MMP9 inhibitors, we first examined MMP9 expression in RPS14-deficient cells. After transduction of human BMCD34+ cells with lentivirus expressing RPS14 shRNAs, we sorted GFP+ cells and observed MMP9 levels by RT-qPCR and Western blot analysis. We found a 4-fold increase in the expression levels of MMP9 in the RPS14 knockdown cells, supporting the hypothesis that MMP9 is increased in RPS14-deficient cells ultimately leading to decreased erythropoiesis.To determine the effects of MMP9 treatment during erythropoiesis, we treated human BMCD34+ cells with recombinant MMP9 protein (rMMP9). Cells were treated with activated rMMP9 for 2 days, and then examined by FACS analysis. Interestingly, we found that rMMP9 treatment decreased the CD71+ erythroid population by 40% compared to vehicle control. We confirmed that enhanced expression of MMP9 is critical to the negative regulation of erythroid development in RPS14 knockdown cells by a performing double knockdown experiment. We co-transduced lentivirus expressing shMMP9-mCherry and shRPS14-GFP shRNAs and examined erythropoiesis by FACS analysis. Our results demonstrated that MMP9 and RPS14 double knockdown partially rescued the decreased CD71+ population in RPS14 knockdown cells. However, MMP9 knockdown alone did not affect CD71+ erythroid development. Taken together, our data suggest that increased MMP9 contributes to the erythroid defect observed in RPS14-deficient cells.In this study, our results showed that MMP9 inhibitors rescued the erythroid defects in RPS14 downregulated del(5q) MDS models. This suggests a novel role for MMP9 in the pathogenesis of del(5q) MDS and MMP9 inhibitors in the treatment of patients with del(5q) MDS. DisclosuresNo relevant conflicts of interest to declare.

Long non-coding RNA profiling of human lymphoid progenitor cells reveals transcriptional divergence of B cell and T cell lineages.

To elucidate the transcriptional landscape that regulates human lymphoid commitment during postnatal life, we used RNA sequencing to assemble the long non-coding transcriptome across human bone marrow and thymic progenitors spanning the earliest stages of B and T lymphoid specification. Over 3000 novel long non-coding RNA genes (lncRNAs) were revealed through the analysis of these rare populations. Lymphoid commitment was characterized by lncRNA expression patterns that were highly stage-specific and more lineage-specific than protein coding patterns. Protein-coding genes co-expressed with neighboring lncRNA genes were enriched for ontologies related to lymphoid differentiation. The exquisite cell-type specificity of global lncRNA expression patterns independently revealed new developmental relationships between the earliest progenitors in the human bone marrow and thymus.

Abstract 3632: Synergistic interaction of auranofin with PARP inhibitors in ATM-proficient mantle cell lymphoma

Abstract Auranofin (AF) (Ridaura®) is an oral, FDA-approved, lipophilic, gold-containing compound that was used for treating arthritis. Its primary mechanism of action is the inhibition of thioredoxin reductase (TRR) activity. It consequently induces oxidative/ER stress and also inhibits Heat shock protein (Hsp) 90 chaperone function. Ataxia-telangiectasia mutated and Rad3-related (ATR), Brca1, Chk1 and DNA-PK are Hsp90 client proteins that participate in DNA repair pathways. Based on our observation that AF induces DNA damage, we evaluated whether treatment with AF also impairs Hsp90-dependent DNA damage repair pathways in Mantle Cell Lymphoma (MCL) cells. It has been reported that ATM mutations are found in about 50% cases of MCL. Earlier studies have shown that ATM mutant MCL cells are more sensitive to poly-ADP ribose polymerase (PARP) inhibitors in vitro and in vivo. We hypothesized that by inhibiting Hsp90-dependent DNA repair pathways (such as the ATR pathway), AF would sensitize ATM-proficient MCL cells to PARP inhibition by ABT-888. We demonstrate that treatment of ATM-proficient MCL cell lines, JeKo-1, MO2058 and Z138C with AF induced reactive oxygen species (ROS) levels and resulted in a concomitant decrease in TRR activity. Microarray analysis of AF-treated primary MCL cells revealed that AF significantly altered heat shock response genes, ER and oxidative stress-inducible genes and a subset of genes that were regulated by ATM or Brca1. Treatment with AF also induced Hsp90 hyperacetylation and resulted in the depletion of Hsp90 client proteins including ATR, AKT and Cyclin D1 in MCL cells. Exposure to AF induced significantly more apoptosis in primary MCL cells, as compared to CD19+ normal B cells, CD34+ human cord blood and bone marrow progenitor cells (< 15% apoptosis) (p < 0.01). Co-treatment of MCL cells with AF and the PARP inhibitor ABT-888 resulted in synergistic increase in apoptosis of ATM-proficient MCL cells, with an increase in the induction of γH2AX foci and depletion of p-Chk1 (a downstream target of ATR signaling). This was accompanied with a concomitant increase in the levels of the ER stress-induced transcription factor CHOP (C/EBP homologous protein), cleaved Caspase 3 and PARP. Collectively, our data suggests the synthetic lethal effects observed by the simultaneous inhibition of DNA repair pathway(s) and PARP activity can be extended to ATM-proficient MCL cells. These pre-clinical studies create a strong rationale to determine the in vivo activity of the combination ABT-888 with AF in MCL. Citation Format: Siddhartha Ganguly, Trisha Home, Sumedha Gunewardena, Scott Weir, Joseph McGuirk, Rekha M. Rao. Synergistic interaction of auranofin with PARP inhibitors in ATM-proficient mantle cell lymphoma. [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 3632. doi:10.1158/1538-7445.AM2015-3632

Human cytomegalovirus reprograms human bone marrow progenitor cells into nitric oxide-producing, long-life, immune suppressive monocyte subset to achieve latency (VIR9P.1142)

Abstract It is generally regarded that human cytomegalovirus (HCMV) achieves latency in human haematopoietic progenitor cells. However, the nature of host cell at HCMV latent infection and the mechanisms governing the differentiation of progenitor cells mediated by HCMV remain elusive. In the present study, we identify that nitric oxide (NO)-producing, long-life, and immune suppressive monocyte subset differentiated from human bone marrow CD34+ progenitor cells serve as the true host for HCMV latency. This monocyte subset can suppress T cell proliferation and activity via NO-based mechanism. Both gain-of-function and lose-of-function assays show that high level NO production in progenitor cells via inducible nitric oxide synthase during HCMV infection is a key to suppress HCMV immediate early (IE) protein expression and viral replication to induce HCMV latency. NO production, correlating with progenitor cell differentiation, is promoted by signal transducer and activator of transcription 3 (STAT3) which activity is increased by HCMV infection. As a control, HCMV strain with US28 deletion, which cannot activate STAT3, fails to produce NO and achieve latency in haematopoietic progenitor cells. Our results demonstrate for the first time that HCMV achieves latent infection through reprograming human haematopoietic progenitor cells into NO-producing, long-life, immune suppressive monocyte subset.

APTO-253 Induces KLF4 to Promote Potent in Vitro Pro-Apoptotic Activity in Hematologic Cancer Cell Lines and Antitumor Efficacy As a Single Agent and in Combination with Azacitidine in Animal Models of Acute Myelogenous Leukemia (AML)

APTO-253, a small molecule that mediates anticancer activity through induction of the Krüppel-like factor 4 (KLF4) tumor suppressor, is being developed clinically for the treatment of acute myelogenous leukemia (AML) and high risk myelodysplastic syndromes (MDS). APTO-253 was well tolerated in a Phase I study in patients with solid tumors using a dosing schedule of days 1, 2, 15, 16 of a 28 day cycle (2T-12B-2T-12B), but recent scientific observations guided APTO-253 toward AML and high risk MDS. Indeed, suppression of KLF4 was reported as a key driver in the leukemogenesis of AML and subsets of other hematologic diseases. The vast majority (~90%) of patients with AML aberrantly express the transcription factor CDX2 in human bone marrow stem and progenitor cells (HSPC) (Scholl et al., J Clin Invest. 2007, 117(4):1037-48). The CDX2 protein binds to CDX2 consensus sequences within the KLF4 promoter, thereby suppressing KLF4 expression in HSPC (Faber et al., J Clin Invest. 2013, 123(1):299-314). Based on these observations, the anticancer activity of APTO-253 was examined in AML and other hematological cancers. APTO-253 showed potent antiproliferative activity in vitro against a panel of blood cancer cell lines, with ηM IC50values in AML (6.9 - 305 ηM), acute lymphoblastic leukemia and chronic myeloid leukemia (39 – 250 ηM), non-Hodgkin’s lymphoma (11 – 190 ηM) and multiple myeloma (72 – 180 ηM). To explore in vivo efficacy, dose scheduling studies were initially conducted in the H226 xenograft model in mice. In the H226 model, APTO-253 showed improved antitumor activity when administered for two consecutive days followed by a five day break from dosing (2T-5B) each week, i.e. on days 1,2, 8,9, 15,16, 22,23, compared to the 2T-12B-2T-12B schedule. The 2T-5B schedule was used to evaluate antitumor activity of APTO-253 in several AML xenograft models in mice. In Kasumi-1 AML and KG-1 AML xenograft models, APTO-253 showed significant antitumor activity (p = 0.028 and p=0.0004, respectively) as a single agent when administered using the 2T-5B schedule each week for four weeks compared to control animals. Mice treated with APTO-253 had no overt toxicity based on clinical observations and body weight measurements. Mice bearing HL-60 AML xenograft tumors were treated with APTO-253 for one day or two consecutive days per week for three weeks, either as a single agent or combined with azacitidine, or with azacitidine alone twice per week (on days 1,4, 8, 11, 15 and 18). APTO-253 as a single agent inhibited growth of HL-60 tumors to approximately the same extent as azacitidine. Furthermore, both once weekly and twice weekly dosing of APTO-253 in combination with azacitidine resulted in significantly enhanced antitumor activity relative to either single agent alone (p = 0.0002 and p = 0.0006 for 1X and 2X weekly APTO-253 treatment, respectively, compared to control). Likewise, using a THP-1 AML xenograft model, APTO-253 administered as a single agent using the 2T-5B per week schedule showed significant efficacy, similar to that of azacitidine, while the combination of APTO-253 and azacitidine demonstrated greatly improved antitumor effects relative to either drug alone. APTO-253 was effective and well tolerated as a single agent or in combination with azacitidine in multiple AML xenograft models, plus APTO-253 does not cause bone marrow suppression in animal models or humans. Taken together, our results indicate that APTO-253 may serve as a targeted agent for single agent use and may provide enhanced efficacy to standard of care chemotherapeutics for AML and other hematological malignancies. DisclosuresRice:Lorus Therapeutics Inc.: Employment. Vellanki:Lorus Therapeutics Inc.: Employment. Lee:Lorus Therapeutics Inc.: Employment. Lightfoot:Lorus Therapeutics Inc.: Employment. Peralta:Lorus Therapeutics Inc.: Employment. Jamerlan:Lorus Therapeutics Inc.: Employment. Jin:Lorus Therapeutics Inc.: Employment. Lum:Lorus Therapeutics Inc.: Employment. Cheng:Lorus Therapeutics Inc.: Employment.

Effect of μ-opioid agonist DAMGO on surface CXCR4 and HIV-1 replication in TF-1 human bone marrow progenitor cells.

BackgroundApproximately one-third of the AIDS cases in the United States have been attributed to the use of injected drugs, frequently involving the abuse of opioids. Consequently, it is critical to address whether opioid use directly contributes to altered susceptibility to HIV-1 beyond the increased risk of exposure. Previous in vitro and in vivo studies addressing the role of μ-opioid agonists in altering levels of the co-receptor CXCR4 and subsequent HIV-1 replication have yielded contrasting results. The bone marrow is believed to be a potential anatomical sanctuary for HIV-1.MethodsThe well-characterized CD34+CD38+ human bone marrow–derived hematopoietic progenitor cell line TF-1 was used as a model to investigate the effects of the μ-opioid receptor–specific peptide DAMGO (D-Ala2,N-Me-Phe4, Gly5-ol-enkephalin) on CXCR4 expression as well as infection of undifferentiated human hematopoietic progenitor cells.ResultsThe results revealed the presence of the μ-opioid receptor-1 isoform (MOR-1) on the surface of TF-1 cells. Furthermore, immunostaining revealed that the majority of TF-1 cells co-express MOR-1 and CXCR4, and a subpopulation of these double-positive cells express the two receptors in overlapping membrane domains. Three subpopulations of TF-1 cells were categorized based on their levels of surface CXCR4 expression, defined as non-, low-, and high-expressing. Flow cytometry indicated that treatment with DAMGO resulted in a shift in the relative proportion of CXCR4+ cells to the low-expressing phenotype. This result correlated with a >3-fold reduction in replication of the X4 HIV-1 strain IIIB, indicating a role for the CXCR4 high-expression subpopulation in sustaining infection within this progenitor cell line.ConclusionsThese experiments provide insight into the impact of μ-opioid exposure with respect to inhibition of viral replication in this human TF-1 bone marrow progenitor cell line model.

Adhesive and mechanical regulation of mesenchymal stem cell differentiation in human bone marrow and periosteum-derived progenitor cells

SummaryIt has previously been demonstrated that cell shape can influence commitment of human bone marrow-derived mesenchymal stem cells (hBMCs) to adipogenic, osteogenic, chondrogenic, and other lineages. Human periosteum-derived cells (hPDCs) exhibit multipotency similar to hBMCs, but hPDCs may offer enhanced potential for osteogenesis and chondrogenesis given their apparent endogenous role in bone and cartilage repair in vivo. Here, we examined whether hPDC differentiation is regulated by adhesive and mechanical cues comparable to that reported for hBMC differentiation. When cultured in the appropriate induction media, hPDCs at high cell seeding density demonstrated enhanced levels of adipogenic or chondrogenic markers as compared with hPDCs at low cell seeding density. Cell seeding density correlated inversely with projected area of cell spreading, and directly limiting cell spreading with micropatterned substrates promoted adipogenesis or chondrogenesis while substrates promoting cell spreading supported osteogenesis. Interestingly, cell seeding density influenced differentiation through both changes in cell shape and non-shape-mediated effects: density-dependent adipogenesis and chondrogenesis were regulated primarily by cell shape whereas non-shape effects strongly influenced osteogenic potential. Inhibition of cytoskeletal contractility by adding the Rho kinase inhibitor Y27632 further enhanced adipogenic differentiation and discouraged osteogenic differentiation of hPDCs. Together, our results suggest that multipotent lineage decisions of hPDCs are impacted by cell adhesive and mechanical cues, though to different extents than hBMCs. Thus, future studies of hPDCs and other primary stem cell populations with clinical potential should consider varying biophysical metrics for more thorough optimization of stem cell differentiation.

Effect of auranofin on oxidative and endoplasmic reticulum stress as well as anti-CLL activity with proteasome inhibitor.

e13568 Background: The gold-containing compound auranofin (AF) (Ridaura), is a treatment of rheumatoid arthritis. Recently, AF was shown to inhibit thioredoxin reductase (TRR), increase reactive oxygen species (ROS) and induce apoptosis in cancer cells. Methods: We determined the ability of AF (250 to 1000 nM) to induce oxidative, proteotoxic and lethal endoplasmic reticulum (ER) stress in CD19+ primary CLL cells, including those with deletion of chromosome 17p. Results: Treatment with AF induced 20 to 40% increase in ROS levels, decreased TRR activity (mean of 45%), but not its protein expression, in CLL cells. AF-mediated oxidative stress induced NRF2 activity with increase in hemeoxygenase-1 (HO-1) and glutamate cysteine ligase modifier (GCLM) levels. AF induced ER stress, associated with increase in GRP78 and the pro-apoptotic transcription factor CHOP protein levels. AF also induced the pro-apoptotic BH3-only domain protein BIM. Exposure to AF increased the intracellular levels of misfolded polyubiquitylated proteins. This disrupted the binding of heat shock protein (hsp) 90 with histone deacetylase 6 (HDAC6), resulting in hyperacetylation and inhibition of the chaperone function of hsp90. This led to proteasomal degradation of CLL-relevant hsp90 client proteins, including ZAP70, c-Raf and AKT. Nuclear STAT3 levels also declined. Exposure to AF induced significantly more apoptosis (range 40 to 60%) in primary CLL cells, as compared to CD19+ normal B cells and CD34+ human cord blood and bone marrow progenitor cells (< 15% apoptosis) (p < 0.01). AF treatment also induced apoptosis in CD19+ cells from patients with poor prognosis CLL with deletion of 17p or with deletion of 13q. Co-treatment with AF and the proteasome inhibitor carfilzomib or the GCLM antagonist buthionine sulfoximine (BSO) synergistically induced apoptosis in primary CLL cells. A FDA IND-supported phase II clinical trial of AF (6 to 9 mg PO/day) with correlative biomarker analysis (as above) is underway and three patients are enrolled. Conclusions: These findings support the full evaluation of clinical activity and predictive biomarkers of response to the re-purposed AF in patients with CLL.

Combinatorial Human Progenitor Cell Transplantation Optimizes Islet Regeneration Through Secretion of Paracrine Factors

Transplanted human bone marrow (BM) and umbilical cord blood (UCB) progenitor cells activate islet-regenerative or revascularization programs depending on the progenitor subtypes administered. Using purification of multiple progenitor subtypes based on a conserved stem cell function, high aldehyde dehydrogenase (ALDH) activity (ALDH(hi)), we have recently shown that transplantation of BM-derived ALDH(hi) progenitors improved systemic hyperglycemia and augmented insulin secretion by increasing islet-associated proliferation and vascularization, without increasing islet number. Conversely, transplantation of culture-expanded multipotent-stromal cells (MSCs) derived from BM ALDH(hi) cells augmented total beta cell mass via formation of beta cell clusters associated with the ductal epithelium, without sustained islet vascularization. To identify paracrine effectors produced by islet-regenerative MSCs, culture-expanded BM ALDH(hi) MSCs were transplanted into streptozotocin-treated nonobese diabetic/severe combine immune deficient (SCID) mice and segregated into islet-regenerative versus nonregenerative cohorts based on hyperglycemia reduction, and subsequently compared for differential production of mRNA and secreted proteins. Regenerative MSCs showed increased expression of matrix metalloproteases, epidermal growth factor receptor (EGFR)-activating ligands, and downstream effectors of Wnt signaling. Regenerative MSC supernatant also contained increased levels of pro-angiogenic versus pro-inflammatory cytokines, and augmented the expansion of ductal epithelial but not beta cells in vitro. Conversely, co-culture with UCB ALDH(hi) cells induced beta cell but not ductal epithelial cell proliferation. Sequential transplantation of MSCs followed by UCB ALDH(hi) cells improved hyperglycemia and glucose tolerance by increasing beta cell mass associated with the ductal epithelium and by augmenting intra-islet capillary densities. Thus, combinatorial human progenitor cell transplantation stimulated both islet-regenerative and revascularization programs. Understanding the progenitor-specific pathways that modulate islet-regenerative and revascularization processes may provide new approaches for diabetes therapy.

Abstract 2078: Treatment with auranofin induces oxidative and lethal endoplasmic reticulum (ER) stress exerting single agent activity against mantle cell lymphoma cells

Abstract Auranofin (AF) (Ridaura®) is an oral, FDA-approved, lipophilic, gold-containing compound used for the treatment of rheumatoid arthritis. In the present studies, we determined for the first time the lethal activity of AF (100 to 1000 nM) and its underlying mechanism(s) in cultured and primary Mantle Cell Lymphoma (MCL) cells. We demonstrate that treatment with AF in JeKo-1 and Z138C cells induced 2 to 3.5 fold increase in reactive oxygen species (ROS) levels (assessed by flow cytometry), decrease in thioredoxin reductase (TRR) activity (mean reduction of 35-40%) without alteration in the expression of TRR. These findings were associated with induction of nuclear factor erythroid 2-related factor 2 (NRF2) activity marked by substantial increase in heme oxygenase-1 (HMOX1) expression. Microarray analysis revealed that AF treatment significantly upregulated ER stress and oxidative stress inducible genes including that of the ER-stress induced pro-apoptotic transcription factor CHOP (CAAT/enhancer binding protein homologous protein), ATF3 (Activated Transcription Factor 3) and Glutamate-cysteine ligase (GCL), modifier subunit (GCLM), the rate-limiting enzyme in the glutathione (GSH) biosynthesis. Exposure to AF induced significantly more apoptosis in primary MCL cells, as compared to CD19+ normal B cells, CD34+ human cord blood and bone marrow progenitor cells (< 15% apoptosis) (p < 0.01). Based on the observations that AF treatment induced oxidative and ER stress, we determined whether exposure to AF increased the intracellular levels of misfolded and polyubiquitylated proteins, which would disrupt the cytosolic complex consisting of the heat shock protein (hsp) 90, heat shock factor (hsf) 1, histone deacetylase 6 (HDAC6) and p97/VCP, resulting in hyperacetylation and inhibition of the chaperone function of hsp90. Indeed, treatment with AF disrupted the association between hsp90 and HDAC6, HSF1 and p97, resulting in hyperacetylation of hsp90 and depletion of the levels of hsp90 client proteins including HDAC6, AKT and Cyclin D1 in MCL cells. Co-treatment of MCL cells with AF and the proteasome inhibitor carfilzomib (CZ) resulted in synergistic apoptosis in cultured MCL cells and significantly more apoptosis in primary MCL cells, than treatment with each agent alone. Based on the induction of GCLM and anti-oxidant response genes following AF treatment, we tested the anti-MCL activity of the combination of AF and the GCL inhibitor Buthionine sulphoximine (BSO), against MCL cells. Co-treatment with BSO and AF resulted in significantly more apoptosis of MCL cells than treatment with each agent alone. Collectively, our data creates a strong rationale for determining the in vivo activity of AF in patients with MCL as a single agent and in combination with other ER and oxidative stress-inducing agents, especially GCL inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2078. doi:1538-7445.AM2012-2078

Treatment with Auranofin Induces Oxidative and Lethal Endoplasmic Reticulum (ER) Stress Exerting Single Agent Activity Against Primary CLL Cells

Abstract 929Auranofin (AF) (Ridaura®) is an oral, FDA-approved, lipophilic, gold-containing compound used for the treatment of rheumatoid arthritis, based upon its anti-inflammatory and immunosuppressive effects. In the present studies, we determined for the first time the lethal activity of AF (250 to 1000 nM) and its underlying mechanism(s) in CD19+ primary CLL cells, including those with del(17p13.1). We demonstrate that treatment with AF induced 20 to 40% increase in reactive oxygen species (ROS) levels (assessed by flow cytometry), decrease in thioredoxin reductase (TRR) activity (mean reduction of 45%) without alteration in the expression of TRR, as determined in the AF treated cell lysates of CLL cells. AF treatment also increased the levels of thioredoxin binding protein 2. These findings were associated with induction of nuclear factor erythroid 2-related factor 2 (NRF2) activity marked by substantial increase in hemeoxygenase-1 (HO-1) levels. Thus, AF treatment perturbed the redox status in CLL cells. Consistent with increased levels of intracellular ROS, AF treatment also induced the markers of ER stress response including the induction of GRP78 and a sustained increase in the levels of the pro-apoptotic transcription factor CHOP (CAAT/enhancer binding protein homologous protein). This was associated with induction of the levels of the pro-death BH3 only domain protein BIM, which is a known mediator of lethal ER stress. Exposure to 1000 nM AF induced significantly more apoptosis (range 40 to 60%) in primary CLL cells, as compared to CD19+ normal B cells and CD34+ human cord blood and bone marrow progenitor cells (< 15% apoptosis) (p < 0.01). AF treatment induced apoptosis to a similar extent in primary CLL cells with del(17p13.1) as compared to other genetic abnormalities. Based on the observations that AF treatment induced oxidative and ER stress, we determined whether exposure to AF also increased the intracellular levels of misfolded and polyubiquitylated proteins, which would disrupt the cytosolic complex consisting of the heat shock protein (hsp) 90, heat shock factor (hsf) 1, histone deacetylase 6 (HDAC6) and p97/VCP, resulting in hyperacetylation and inhibition of the chaperone function of hsp90. Indeed, treatment with AF disrupted the association between hsp90 and HDAC6, resulting in hyperacetylation of hsp90 and depletion of the levels of HDAC6 in CLL cells. AF mediated hyperacetylation of hsp90 and α-tubulin was associated with proteasomal degradation of several of the CLL-relevant hsp90 client proteins, including ZAP70, c-Raf and AKT. Co-treatment of CLL cells with AF and the proteasome inhibitor carfilzomib (20 nM) significantly restored the levels of ZAP70, c-Raf, AKT and HDAC6. Finally, relatively lower levels of AF (250 nM) combined with the pan-HDAC inhibitor panobinostat (20 nM) or carfilzomib (5 nM) induced more cell death of primary CLL cells than each agent alone. These findings indicate that treatment of primary CLL cells with AF results in oxidative and ER stress. AF treatment also promotes the depletion of pro-survival CLL-relevant hsp90 client proteins. Collectively, our data creates a strong rationale for determining the in vivo activity of AF in patients with CLL including those with del(17p13.1). Disclosures:Reyes:Millennium, Sanofi Aventis: Consultancy.

Dexamethasone and lenalidomide have distinct functional effects on erythropoiesis

AbstractCorticosteroids and lenalidomide decrease red blood cell transfusion dependence in patients with Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome (MDS), respectively. We explored the effects of dexamethasone and lenalidomide, individually and in combination, on the differentiation of primary human bone marrow progenitor cells in vitro. Both agents promote erythropoiesis, increasing the absolute number of erythroid cells produced from normal CD34+ cells and from CD34+ cells with the types of ribosome dysfunction found in DBA and del(5q) MDS. However, the drugs had distinct effects on the production of erythroid progenitor colonies; dexamethasone selectively increased the number of burst-forming units-erythroid (BFU-E), whereas lenalidomide specifically increased colony-forming unit-erythroid (CFU-E). Use of the drugs in combination demonstrated that their effects are not redundant. In addition, dexamethasone and lenalidomide induced distinct gene-expression profiles. In coculture experiments, we examined the role of the microenvironment in response to both drugs and found that the presence of macrophages, the central cells in erythroblastic islands, accentuated the effects of both agents. Our findings indicate that dexamethasone and lenalidomide promote different stages of erythropoiesis and support the potential clinical utility of combination therapy for patients with bone marrow failure.