Bone Marrow Colony-forming Units Research Articles

Abstract C132: SGN-35T: A novel CD30-directed antibody-drug conjugate for the treatment of lymphomas

Abstract Antibody-drug conjugates (ADCs) employing the vedotin drug linker are effective anti-cancer agents in multiple indications including Hodgkin lymphoma, cervical cancer, and bladder cancer. While vedotin ADCs have demonstrated efficacy for the treatment of a wide variety of solid and hematologic cancers, some side effects, including dose-limiting neutropenia, are commonly observed. To improve the tolerability while leveraging the known activity of brentuximab vedotin, an ADC approved for the treatment of advanced classical Hodgkin lymphoma and other CD30-expressing lymphomas, alternative ADC structural components are under exploration. A novel cleavable linker containing a tripeptide molecule comprised of D-leucine-alanine-glutamate (DLAE) was developed and used to generate SGN-35T, an anti-CD30 ADC with the same mAb backbone and cytotoxic monomethyl auristatin E (MMAE) payload as brentuximab vedotin. SGN-35T was investigated for both anti-tumor activity and effects on hematopoiesis using in vitro and in vivo assays. SGN-35T displayed comparable activity to brentuximab vedotin against three lymphoma cell lines in vitro, and complete or near-complete tumor growth inhibition in five lymphoma xenograft models including a CD30-heterogeneous model. SGN-35T led to cell cycle arrest at the G2/M phase leading to apoptosis, in concordance with the established mechanism of action of MMAE. Introduction of the tripeptide linker in SGN-35T maintained the induction of immunogenic cell death hallmarks observed with brentuximab vedotin and other vedotin-based ADCs. SGN-35T demonstrated reduced potency on hematopoiesis compared to brentuximab vedotin in a human bone marrow colony-forming unit assay. In cynomolgus monkey toxicity studies, administration of SGN-35T had a reduced impact to neutrophils counts at matched dose levels and a two-fold improvement in tolerability compared to brentuximab vedotin, with a highest non-severely toxic dose (HNSTD) of 6 mg/kg versus an HNSTD of 3 mg/kg for brentuximab vedotin when administered Q3Wx4. Together, these data demonstrate that SGN-35T exhibits anti-tumor activity in preclinical models and may reduce the severity of hematopoietic toxicities while retaining anti-tumor activity observed with brentuximab vedotin. A Phase 1, first-in-human study is planned to evaluate the safety and anti-tumor activity of SGN-35T in lymphoid malignancies. Citation Format: Kevin J Hamblett, Steven Jin, Roma Yumul, Yufei Chen, John Kwon, Jessica Simmons, Clark Henderson, Hao Sun, Jason P Schrum, Andrea R Lim, Noah A Bindman, Melissa Conerly, Astrid Clarke. SGN-35T: A novel CD30-directed antibody-drug conjugate for the treatment of lymphomas [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C132.

Dysregulation of immune cell and cytokine signalling correlates with clinical outcomes in myelodysplastic syndrome (MDS).

Myelodysplastic syndromes (MDS) are characterised by ineffective haematopoiesis. Although hypomethylating agents (HMA) have improved survival in higher-risk MDS, most patients eventually succumb to progressive disease. Utilising samples collected prospectively from three MDS clinical trials, we analysed genetic and immunological biomarkers and correlated them with clinical outcomes. A hundred and fifty four samples were analysed from 133 de novo MDS patients for T-cell and myeloid cell immunophenotyping and gene expression analysis. Treatments were with HMA or immunomodulatory drug (IMiD) alone or in combination. We observed differences in immune cell subsets between lower- and higher-risk IPSS groups with NKT cells, MDSCs, intermediate-proinflammatory and non-classical monocytes being higher in the latter group, while naïve CD4+ T cells were reduced. Intermediate-proinflammatory monocytes were increased in non-responders and those failing to achieve at least a haematological improvement. Proinflammatory NKT cells were increased at diagnosis for patients failing to derive clinical benefit after 12months of treatment. Gene expression analysis of paired bone marrow (BM) colony-forming units (CFUs) from diagnosis and 4 cycles post-treatment confirmed that genes involved in cytokine signalling were downregulated in C4 normal colonies. These findings support the central roles of dysregulation in innate immunity and inflammatory signalling in the pathogenesis of MDS which correlated with clinical outcomes post-treatment.

Abstract 1548: Genetic biomarkers predict clinical response and survival in myelodysplasia

Abstract Background: Hypomethylating agents (HMA) used in higher-risk myelodysplastic syndromes (MDS) improve survival but HMA-failure has a poor prognosis. Abnormal bone marrow (BM) colony-forming units (CFUs) persist in treated MDS patients despite achievement of complete remission, suggesting persistent abnormal stem cell function. We aim to identify genetic biomarkers following treatment with Azacitidine ± Thalidomide or Lenalidomide that predict clinical outcomes in MDS. Methods: BM cells from patients enrolled in ALLG MDS3 and MDS4 clinical trials at baseline and after 4 cycles of treatment (C4) were grown in Methocult for 14 days. CFUs were pooled at baseline; C4 macroscopically normal and abnormal colonies were harvested separately. mRNA expression was quantified using the Nanostring nCounter PanCancer Pathways panel. Clinical outcomes analysed were: (1) clinical benefit at 12 months (haematological improvement or better as per IWG criteria) (2) best response achieved. Genes expressed above background level in ≥25% of samples were included for statistical analyses, resulting in 516 genes across 56 samples from 23 different patients. R limma package was used for differential expression analysis. Patients were weighted using limma's voomWithQualityWeights function. Moderated t-tests with empirical Bayes were done to identify differentially expressed genes. For testing between colonies, a log-fold-change cut-off of 0.5 was used with limma's treat function. P-values were adjusted for multiple hypothesis testing. Results: 98 genes exhibited significantly different expression (p <0.05) when comparing C4 normal to baseline colonies and 118 genes were differentially expressed between C4 normal to C4 abnormal colonies. Key results are summarised: (1) Within C4 normal colonies - RFC3 (p=0.04) and LTBP1 (p=0.04) were upregulated in patients with clinical benefit at 12 months (2) Within C4 abnormal colonies - FN1 (p=0.03) was upregulated in those failing to achieve at least a partial response (PR) (3) Comparing C4 normal colonies to baseline: a. The top 5 differentially expressed genes: MAPK12, PLAU, FGFR1, IL10 and FLNA (p<0.001) were downregulated in the C4 normal group; b. In addition, for patients with clinical benefit, MYD88 and PIK3R5 were downregulated (p<0.001) while; c. In patients who achieved at least a PR as best response, NFKB1, SYK and TGFBR2 were downregulated (p<0.001). d. Gene ontology analysis revealed upregulated genes involved in plasma membrane and cytokine production were over-represented at baseline while; e. KEGG pathway analysis showed upregulated genes involved in the cytokine receptor, Notch and NF-kB signalling pathways. Conclusion: We identified changes in gene expression following treatment in MDS that predict outcomes in response and clinical benefit. These genetic biomarkers require further validation and could define early markers of resistance for investigation of novel therapies. Citation Format: Lynette Chee, David Ritchie, Jessica Chung, Daniel Park, Mandy Ludford-Menting, Jane Ripley, Melita Kenealy, Rachel Koldej. Genetic biomarkers predict clinical response and survival in myelodysplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1548.

MATHEMATICAL ANALYSIS OF FUNCTIONAL PROPERTIES OF MICE BONE MARROW IN THE INITIAL PHASE OF ACUTE FRACTIONATED IRRADIATION.

to determine the quantitative characteristics of population functioning of mice bone marrow colony-forming units during seven days of acute fractionated irradiation. Assigned task is solved by means of described in works R. V. Boiko et al. (2015, 2016) math-ematical model of alterations in the number of bone marrow colony-forming units using the experimental results ofwork W.Chu-Tse, L. G.Lajtha (1975). Mathematical model is developed basing on the new hematopoiesis scheme,which was introduced by I. Chertkov(1984, 1991). By applying original mathematical model of hematopoiesis scheme using results concerning the change innumber of bone marrow colony-forming units of mice femur we determined quantitative characteristics of theirfunctioning during seven days of fractionated irradiation under daily acute γ-radiation in the dose of 0.7 Gy. Mathematical model is introduced, which describes changes in the relative number of colony-formingunits in mice bone marrow in the process of their acute fractionated irradiation.

A Preclinical Non-Human Primate Model to Test Reversible Myeloablation Following IV Busulfan Therapy without Stem Cell Rescue

Busulfan is a standard drug utilized in stem cell transplantation for myeloid malignancies, however it is not used in any induction or salvage chemotherapy regimen in these diseases. Non-human primates (NHP) represent a useful model for preclinical evaluation of chemotherapy-related toxicity. In order to identify what dose of IV busulfan could be tested in clinical studies for acute myeloid leukemia, we utilized NHP to determine the highest dose of IV busulfan needed to achieve reversible myelosuppression in unperturbed bone marrow (BM). Nine adult baboons (Papio anubis) were divided into three groups (n=3/group) for this study. Group A received the lowest dose of busulfan 6.4 mg/kg (1.6 mg/kg/day x 4 days), Group B received 8 mg/kg (3.2 mg/kg on day 1 and 1.6 mg/kg on day 2-4) and Group C received 9.6 mg/kg (3.2 mg/kg/day day 1-2 and 1.6 mg/kg/day day 3-4). Peripheral blood (PB) complete blood count (CBC), and BM CD34+ cells, colony forming unit (CFU) were monitored over 90 days after busulfan. Maximum suppression of WBC count, hemoglobin (Hb) and platelet counts in the PB of baboons in all 3 groups were observed around day 15. The suppression of WBC was 57 ± 4%, 58 ± 4% and 53 ±10% in group A, B and C respectively (group A: p = 0.006, group B: p = 0.03, group C: p = 0.07). The suppression of absolute neutrophil counts (ANC) in group A, B and C were 85 ± 2%, 8 4± 1% and 82± 5% respectively. Similarly the minimum post-busulfan Hb levels were 10± 0.6 g/dl, 10± 1g/dl, 10± 0.6g/dl in group A, B and C respectively. The suppression of platelet counts were 84 ± 6%, 81 ± 7% and 81 ± 9% in group A, B and C respectively. Taken together, PB CBC results indicate that at day 15, despite administering a higher dose of busulfan the maximum depletion of ANC, Hb and platelet was comparable among the three groups. At day 90, the PB WBC, Hb and platelet counts returned within normal ranges. Notably, in the group receiving the highest dose of busulfan (Group C) the PB WBC counts at day 90 displayed 76 ± 9% recovery and platelets a 79 ± 13% recovery. Extra-hematopoietic toxicity, including weight loss, was not significant in any group of animals. Then we examined CD34+ cells and CFU content of BM prior to and following busulfan therapy. Baboons receiving the lowest dose of busulfan (Group A) displayed 75 ± 19% CD34+ cell suppression, while the intermediate group B had 90 ± 9%, and the group C which received the highest dose of busulfan had 98 ± 0.6 % suppression. The CD34+ cell recovery at day 90 was 56%, 35% and 24% in group A, B and C, respectively, suggesting that the BM CD34+ cell compartment requires more time to recover despite near normal CBC numbers. Similarly, at day 15 the suppression of BM CFU in group A, B and C was 71 ± 10% , 86 ± 8% and 91 ± 5% respectively. Although the suppression in PB WBC count was not distinct the absolute number of CD34+ cells and CFU content of BM varied based on busulfan dose administered. At day 90 the recovery of BM CFU in group A, B & C were 51 ± 15% , 79 ± 15% and 53 ± 9%, respectively. Although there was no significant intergroup difference in degree of CFU plating efficiency, pre-busulfan versus day 90 CFU recovery in group C was statistically significant (p = 0.02) . These results further validate the hierarchy of CD34+ cells and CFU in contrast to more mature PB cells and the cell populations targeted by busulfan, emphasizing regulatory systems governing homeostasis are likely maintained by reserved BM precursors and progenitor cells. Despite near normalization of blood counts, the relatively primitive BM CD34+ cells and CFU content require longer time to return to pre treatment levels. BM biopsy results indicate a return of cellularity and tri-lineage hematopoiesis comparable to pre-treatment levels in all three groups by day 90. Taken together, our study showed that when busulfan is administered in a dose range of 6.4 to 9.6 mg/kg in an NHP model, it is capable of inducing reversible myeloablation with tolerable toxicity without requiring stem cell rescue or blood transfusions. Our results also indicate that about 30 to 40% BM reserve is capable of maintaining normal PB cell counts. Based on the results, we plan to design a phase 1/2 clinical trial where non-myeloablative doses of IV busulfan will be tested as salvage therapy in patients with acute myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

Abstract C52: PARP1 trapping activity of PARP inhibitors is associated with cytotoxicity in both cancer cells and healthy bone marrow

Abstract Background: PARP inhibitors (PARPi) are under clinical investigation as anticancer therapies in several indications with a focus on monotherapy in tumors with homologous recombination deficits and on combination regimens with DNA damaging chemotherapy. The combination activity of PARPi with DNA alkylating agents is due in part to trapping of PARP1 onto base excision repair intermediates. Recent studies in mice indicate that potent trapping activity limits the tolerability of PARPi in this setting. While trapping has not been directly demonstrated with PARP inhibitors alone, genetic and correlative data suggest that it may be important in the monotherapy setting as well. The objective of this study was to further delineate the role of trapping in single-agent cytotoxicity of PARPi in cancer cells and healthy bone marrow. Methods: Cytotoxic potency was determined for cancer cells using CellTiter-Glo and for erythroid and myeloid progenitors using colony forming unit (CFU) assays. PARP1 trapping was evaluated by cellular fractionation, TR-FRET and BLI. PARPi binding affinities and off-rates were determined via SPR. Cellular PAR levels were determined by ELISA. Results: Talazoparib, rucaparib and the novel PARPi A-934935 bound to PARP1 with similar affinities and off-rates and inhibited cellular PAR synthesis with identical potencies. Despite these similarities, talazoparib was a more potent trapping agent. Consistent with the increased trapping activity, talazoparib was >10-fold more potent than rucaparib and A-934935 at inhibiting proliferation of erythroid progenitors and >50-fold more potent in myeloid progenitors. Similar differences were observed in a panel of human cancer cell lines including BRCA−/− breast cancer and small cell lung cancer. Conclusions: All NAD+-competitive PARPi elicit PARP1 trapping, but different inhibitors display varying degrees of resolution between trapping and cellular PAR depletion. Understanding the relative importance of these mechanisms to the tolerability and efficacy of PARP inhibitors is of critical importance. Myelosuppression is a commonly observed toxicity in PARPi clinical trials. In the present study we used ex vivo bone marrow CFU assays to model myelosuppression. The IC50s observed were within the range of exposures observed at recommended phase II doses in monotherapy clinical trials, highlighting the clinical relevance of this experimental approach. Our data reveal that compounds inhibiting cellular PAR synthesis with equal potency span a broad range of potency in BM-CFU assays in a manner predicted by PARP1 trapping activity. These results suggest that PARP1 trapping is a primary driver of myelosuppression by PARP inhibitors. Our observation that trapping drives cytotoxicity to a similar extent in bone marrow and cancer cells sensitive to single-agent PARP inhibition suggests that PARP1 trapping activity is not advantageous with respect to the therapeutic index of PARPi in a monotherapy setting. This is consistent with observations related to the therapeutic index of PARP inhibitors in the clinic. These results also have important implications for selection of a PARPi for combination regimens where trapping is not required for efficacy and in which myelosuppression may be a concern, such as those containing platinum drugs or topoisomerase I inhibitors. Citation Format: Todd A. Hopkins, Julie L. Wilsbacher, Enrico L. DiGiammarino, Sanjay C. Panchal, Gui-Dong Zhu, Thomas D. Penning, Eric F. Johnson, David Maag. PARP1 trapping activity of PARP inhibitors is associated with cytotoxicity in both cancer cells and healthy bone marrow. [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 C52.

Autophagy Is Required For Long-Term Hematopoietic Stem Cell (HSC) Function and G-CSF-Induced HSC Mobilization

Hematopoiesis originates from a rare pool of hematopoietic stem cells (HSC) that are uniquely capable of both self-renewal and terminal differentiation into lineage-committed progenitor cells. Autophagy is a process of cytoplasmic protein recycling which maintains cellular homeostasis and protects the cell during periods of metabolic stress and nutrient deprivation and has an established role in the survival and function of immunological cells. Recent publications have linked autophagy with preservation of normal long term HSCs (LT-HSCs) during aging (Nature; 2013: 494:323-7). We therefore sought to determine the role of autophagy in LT-HSC function at homeostasis and during the clinically relevant stress of G-CSF-induced HSC mobilization.Using single cell imaging flow cytometry to monitor autophagosome formation in LC3-GFP transgenic mice (LC3-GFP punctae formation is a reporter of autophagy activity) we demonstrated autophagic activity in HSC populations but not in committed myeloid progenitors. In line with this, inhibition of autophagy degradation by chloroquine administration resulted in the accumulation of autophagy related protein p62 in purified HSCs compared to myeloid progenitors. To determine the contribution of autophagy to HSC development and function, we analyzed mice deficient in Atg5, a protein essential for autophagosome formation. Autophagy was not required for fetal liver (FL) HSC development, however, Atg5-/- FL HSCs showed mildly reduced long-term repopulating function in bone marrow (BM) transplantation assays (16 weeks peripheral blood (PB) engraftment Atg5-/- 83.45% vs. WT 93.10%, n=12, p<0.001). Importantly, Atg5-/- LT-HSCs (Lin-Sca-1+c-kit+Flk2-CD150+CD48-) were markedly reduced in congenic recipients compared to WT FL LT-HSCs (Atg5-/- 0.02% vs. WT 0.04%, n=11, p<0.001). Secondary competitive transplantation was used to determine the effect of autophagy loss on LT-HSC function in vivo. Atg5-/- LT-HSCs exhibited a profound impairment in the repopulation of secondary congenic recipients (Atg5-/-4.65% vs. WT 32.3%, n=5, p<0.01).Mechanistically, microarray analysis of purified LT-HSCs from Atg5-/- vs. WT FL chimeras demonstrated clear differences in gene expression by unsupervised hierarchical clustering. Differentially expressed genes included Cxcl12, Sdc2 and Apex1, regulators of HSC fate. Gene Ontology enrichment analysis demonstrated that autophagy deficient LT-HSCs had impaired metabolism, enhanced cellular differentiation, enforced proliferation and increased apoptosis. Validating these findings, there was a loss of quiescence in the Atg5-/- compared to WT LT-HSC (quiescent Atg5-/- 30% vs. WT 39%, n=16 p<0.05) and Atg5-/- FL HSCs exhibited enhanced apoptosis after culture in cytokine enriched media (Atg5-/-21.2% vs. WT 14.2%, n=5, p<0.01).Given the requirement for autophagy in homeostasis of LT-HSC and its role in proliferation and metabolic stress, we next investigated whether autophagy participated in the HSC response to G-CSF. G-CSF is commonly used to ameliorate neutropenia in patients treated with chemotherapy and is also used to mobilize HSCs for patients undergoing HSC transplantation. Using single cell imaging flow cytometry, autophagosome formation was enhanced in HSCs after 6 days of G-CSF mobilization (10mcg/day). G-CSF treatment efficiently mobilized PB neutrophils and colony forming units (CFU) in WT chimeras, however Atg5-/- chimeras showed a striking reduction in G-CSF-induced neutrophil (Atg5-/- 4.95 x106/mL vs. WT 10.46 x106/mL, n=20, p<0.0001) and PB CFU mobilization post-G-CSF (Atg5-/- 47/100µL vs. WT 126/100µL, n=5, p<0.01). BM CFU numbers were similar in Atg5-/- and WT FL chimeras both pre- and post-G-CSF mobilization. Atg5-/-neutrophils demonstrated increased apoptosis after G-CSF treatment suggesting that autophagy limits PB neutrophil survival, but not BM neutrophil development during G-CSF mobilization.These data demonstrate that autophagy is an active process in LT-HSCs and that genetic deletion of Atg5 results in the failure of adult LT-HSC maintenance and function. Autophagic activity is augmented by G-CSF-induced HSC stress and is required for G-CSF-induced HSC mobilization. These findings are particularly relevant to HSC transplantation and hematopoietic function in the context of a rapid rise in the clinical use of agents that have modulatory effects on autophagy. Disclosures:No relevant conflicts of interest to declare.

Hematopoietic G‐protein‐coupled receptor kinase 2 deficiency decreases atherosclerotic lesion formation in LDL receptor‐knockout mice

Leukocyte chemotaxis is deemed instrumental in initiation and progression of atherosclerosis. It is mediated by G-protein-coupled receptors (e.g., CCR2 and CCR5), the activity of which is controlled by G-protein-coupled receptor kinases (GRKs). In this study, we analyzed the effect of hematopoietic deficiency of a potent regulator kinase of chemotaxis (GRK2) on atherogenesis. LDL receptor-deficient (LDLr(-/-)) mice with heterozygous hematopoietic GRK2 deficiency, generated by bone marrow transplantation (n=15), displayed a dramatic attenuation of plaque development, with 79% reduction in necrotic core and increased macrophage content. Circulating monocytes decreased and granulocytes increased in GRK2(+/-) chimeras, which could be attributed to diminished granulocyte colony-forming units in bone marrow. Collectively, these data pointed to myeloid cells as major mediators of the impaired atherogenic response in GRK2(+/-) chimeras. LDLr(-/-) mice with macrophage/granulocyte-specific GRK2 deficiency (LysM-Cre GRK2(flox/flox); n=8) failed to mimic the aforementioned phenotype, acquitting these cells as major responsible subsets for GRK2 deficiency-associated atheroprotection. To conclude, even partial hematopoietic GRK2 deficiency prevents atherosclerotic lesion progression beyond the fatty streak stage, identifying hematopoietic GRK2 as a potential target for intervention in atherosclerosis.

Maternal administration of busulfan before in utero transplantation of human hematopoietic stem cells enhances engraftments in sheep

In utero transplantation (IUT) of human hematopoietic stem cells has been conducted in sheep, which are used as large animal models of human hematopoietic reconstitution and models for clinical IUT; however, the levels of engraftment have generally been low. Busulfan (BU), a myeloablative agent, is often administered to patients before hematopoietic stem cells transplantation to improve the engraftment. In this study, hematopoietic activity was evaluated in adult sheep after administering BU at different doses. Next, pregnant ewes were administered BU, and dams as well as their fetuses were evaluated, as BU readily crosses the sheep placenta. Then, the BU dose with the desired outcomes was selected and administered to pregnant ewes at 2 or 6 days before performing IUT using human cord blood CD34(+) cells. The engraftment was evaluated in recipients that underwent IUT in the presence or absence of BU. As a result, hematopoietic activity was safely and transiently suppressed in adult sheep treated with 5 to 7.5 mg/kg BU. BU crossed the sheep placenta, and fetal sheep were indeed conditioned by administering 3 mg/kg BU to pregnant ewes. Engraftment of human CD34(+) cells in fetal recipients was enhanced when IUT was carried out 6 days post-BU. Up to 3.3% engraftment levels (in terms of bone marrow colony-forming units) were achieved with the IUT of 0.72 to 2.4 million CD34(+) cells when BU was used. BU can be administered to pregnant ewes to effectively condition the fetal recipient for IUT with enhanced engraftment of donor cells.

A p210 BCR/ABL Mutant That Lacks Guanine Nucleotide Exchange Factor Activity Induces Erythroleukemia in a Murine Bone Marrow Transplantation Model,

Abstract 3756The p210 BCR/ABL and p190 BCR/ABL fusion proteins are constitutively active tyrosine kinases that are associated with chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) respectively. Whereas both of these fusion proteins contain equivalent contributions from ABL, they differ in the amount of BCR sequence at the NH2-terminus. We have identified a domain with guanine nucleotide exchange factor (GEF) activity which is contained within the BCR sequences of p210 BCR/ABL that distinguishes it from p190 BCR/ABL. This activity is subject to autoinhibition in the context of BCR, but is constitutively active in p210 BCR/ABL, and cannot be blocked using the tyrosine-kinase inhibitor, Imatinib mesylate. The activities of small GTPases are regulated by GEFs, and are essential for the regulation of hematopoietic stem cell differentiation and proliferation. In a human Ph-positive erythroleukemia cell line (K562), inhibiting p210 BCR/ABL-mediated Ras activation induces erythroid differentiation, while abnormal activation of Cdc42 reduces erythroid progenitors and erythroid colony-forming units in bone marrow. In order to directly investigate the contribution of the GEF activity to p210 BCR/ABL-mediated leukemogenesis, we have introduced a point mutation into p210 BCR/ABL (p210 BCR/ABL(S509A)) that eliminates the activity. The mutation has no discernable effect on tyrosine kinase activity, which is consistent with previous data that the two activities are functionally independent. We have compared p190 BCR/ABL, p210 BCR/ABL, and p210 BCR/ABL(S509A) in a murine bone marrow transplantation (BMT) model. Although all transplanted mice develop splenomegaly with leukocytosis, the p190 BCR/ABL and p210 BCR/ABL(S509A) transplanted mice exhibit a more rapid onset of disease than mice transplanted with p210 BCR/ABL (15–18 days vs 23–38 days). This difference is observed regardless of whether the donor mice are pre-treated with 5-Fluorouracil. The more rapid onset of disease induced by p190 BCR/ABL and p210 BCR/ABL(S509A) is associated with reticulocytosis, and increased numbers of nucleated red blood cells (nRBC), but no anemia. The nRBC/100 WBC counts are 0, 4.75±2.21, 277.5±190.6, and 119.5±66.0 for MIG, p210 BCR/ABL, p190 BCR/ABL and p210 BCR/ABL (S509A) respectively, thus suggesting leukemic expansion of the erythroid lineage. To confirm this difference in lineage expansion, clonogenicity assays were performed. Consistent with previous reports, p210 BCR/ABL supports the growth of CFU G/M, but not BFU-E, and colony growth is completely inhibited by Nilotinib. In contrast, p190 BCR/ABL and p210 BCR/ABL(S509A) support the growth of BFU-E (but not CFU G/M), and these colonies are completely insensitive to Nilotinib treatment. In summary, our observations suggest that a point mutation that eliminates the GEF activity of p210 BCR/ABL produces a phenocopy of p190 BCR/ABL, without affecting the tyrosine kinase activity. These results support a model in which the GEF activity that distinguishes p210 BCR/ABL from p190 BCR/ABL actively regulates disease progression by determining lineage-specific leukemic expansion. Disclosures:No relevant conflicts of interest to declare.

Genz-644282, a Novel Non-Camptothecin Topoisomerase I Inhibitor for Cancer Treatment

Genz-644282 [8,9-dimethoxy-5-(2-N-methylaminoethyl)-2,3-methylenedioxy-5H-dibenzo[c,h][1,6]naphthyridin-6-one] has emerged as a promising candidate for antitumor agents. This report describes the bone marrow colony-forming unit, granulocyte macrophage (CFU-GM) and tumor cell CFU activity of topoisomerase I (Top1) inhibitors, such as Genz-644282, topotecan, irinotecan/SN-38, and ARC-111, and examines their activity in several human tumor xenograft models. Colony-forming assays were conducted with mouse and human bone marrow and eight human tumor cell lines. In addition, 29 human tumor cell lines representing a range of histology and potential resistance mechanisms were assayed for sensitivity to Genz-644282 in a 72-hour exposure assay. The efficacy of Genz-644282 was compared with standard anticancer drugs (i.e., irinotecan, docetaxel, and dacarbazine) in human tumor xenografts of colon cancer, renal cell carcinoma, non-small cell lung cancer, and melanoma. Human bone marrow CFU-GM was more sensitive to the Top1 inhibitors than was mouse bone marrow CFU-GM. The ratio of mouse to human IC(90) values was more than 10 for the camptothecins and less than 10 for Genz-644282, which had more potency as a cytotoxic agent toward human tumor cells in culture than the camptothecins in the colony-forming and 72-hour proliferation assays. Genz-644282 has superior or equal antitumor activity in the human tumor xenografts than the standard drug comparators. On the basis of preclinical activity and safety, Genz-644282 was selected for development and is currently undergoing phase 1 clinical trial.

Characterization of Bone Marrow (BM)- and Fat Tissue-Derived (F) Mesenchymal Stem Cells (MSC) From Patients without Bone Marrow Involvement.

AbstractAbstract 3856Cell therapy with MSCs appears to be a promising method for treatment of different disorders. Bone marrow and fat tissue are both considered as a prospective source of MSCs for therapeutic applications. However, the differences in functional properties of specific populations of MSC derived from these two tissues of the same patient are still poorly investigated.Patients and methods: 23 cardiovasular patients and 4 healthy donors were involved in this study. All patients were enrolled in programme funded by EU FP7. MSC cultures from BM (BM-MSC) and subcutaneous adipose (F-MSC) of the same patient were evaluated in successive passages for immunophenotype (FACS analysis), frequency of colony-forming units (CFU) in MSC population, frequency of adipo- and osteo-progenitors (CFU-Ad, CFU-Ost) in the same population and for dynamic of changes in these properties with successive passage. CFU, CFU-Ad and CFU-Ost were studied by limiting dilution assay followed by induction of adipo- or osteo- differentiation as described (Mitchell et al. Stem Cells 2006) with some modifications. Cell suspension was serially diluted two folds across the 8 columns of 96-well plates, resulting in columns containing from 50 to 0,39 cells per well. After 10 days of culture the number of positive and negative wells was determined for each cell concentration and CFU frequency was calculated. Then plates were induced to undergo adipogenesis and osteogenesis and CFU-Ad was determined by Oil Red staining and CFU-Ost by Alizarin Red staining after 14 and 21 days respectively. All calculations were performed as for CFU. Results.Significant difference was observed in immunophenotype of MSC derived from different tissues of the same donor: while both BM- and F-MSC were positive for stromal cell-associated markers CD105, CD90 and CD73 and were negative for hematopoietic lineage cells markers CD34, CD19, CD14, CD45, the population of CD146+ cells was more abundant in BM-MSC than in F-MSC at passage 1 (39,6%± 8,3% vs 5,6 %± 1,8%; p<0,005), this population of cells further declined in successive passages by passage 5 (17,7%± 6,9% in BM-MSC vs 1,5%± 0,65% in F-MSC; p<0,01); (Figure 1). To further characterize MSC derived from BM and F we studied the frequency of CFU at passages P1-P4, and CFU-Ad and CFU-Ost at passages 2 and 4. Results are shown in Tables 1 and 2. [Display omitted] Table 1Frequency of CFU at successive passages in BM-MSC and F-MSCAssayP1P2P3P4BM-MSCCFU30,6%±4,9(n=10)23,5%±4,0 (n=8)15,3%± 3,7 (n=6)8,0%±1,5 (n=9)F-MSCCFU13,54%±1,7 (n=8)19,1%±2,8 (n=14)21,9%±3,3 (n=14)28,6%±3,0 (n=14)These data support the idea that in vitro expanded BM- and F- derived MSC differ in their properties. While the frequency of CFU in BM derived MSC population declined by as much as 4-fold by passage 4, there was 2-fold increase in frequency of CFU in Ad-MSC over the same period of culturing and this frequency remain unchanged up to passage 7 (data not shown). These data are consistent with our observation that cultures of BM-MSC showed first signs of senescence (senescence-associated β-galactosidase activity) as early as at passage 4,3±0.5 while the first signs of senescence in Ad-MSC cultures were seen at passage 6,67±0,66 (p<0,02). We assume that early decline in CFU frequency in BM-MSC in vitro is because of cell senescence. Importantly, the difference in CFU-Ad and CFU-Ost was discovered: frequency of both declined significantly in BM-MSC by passage 4, while frequency of Ad- and Ost- lineage progenitors remain unchanged in F-MSC cultures. Conclusion:For a first time a broad study was performed to compare MSC derived from BM and adipose tissues for immunophenotype, self-renewal and differentiation potential. We have found that BM-MSC and F-MSC differ significantly in all these characteristics. Differences in BM- and F-MSC could be explained by their ontogeny and/or different microenvironment in “parent” tissue. These variations could affect their efficacy in different therapeutic applications. Disclosures:No relevant conflicts of interest to declare.

Reduction of Radiation-Induced Vascular Nitrosative Stress by the Vitamin E Analog γ-Tocotrienol: Evidence of a Role for Tetrahydrobiopterin

The vitamin E analog γ-tocotrienol (GT3) is a powerful radioprotector. GT3 reduces postradiation vascular peroxynitrite production, an effect dependent on inhibition of hydroxy-methylglutaryl-coenzyme A reductase. Hydroxy-methylglutaryl-coenzyme A reductase inhibitors mediate their pleiotropic effects via endothelial nitric oxide synthase that requires the cofactor tetrahydrobiopterin (BH4). This study investigated the effects of radiation on BH4 bioavailability and of GT3 on BH4 metabolism. Mice were exposed to 8.5 Gy of total body irradiation (TBI). Lung BH4 and total biopterin concentrations were measured 0, 3.5, 7, 14, and 21 days after TBI by use of differential oxidation followed by high-performance liquid chromatography. The effect of exogenous GT3 and BH4 treatment on postradiation vascular oxidative stress and bone marrow colony-forming units were assessed in vivo. The effect of GT3 on endothelial cell apoptosis and endothelial expression of guanosine triphosphate (GTP) cyclohydrolase 1 (GTPCH), GTPCH feedback regulatory protein (GFRP), GFRP transcription, GFRP protein levels, and GFRP-GTPCH protein binding was determined in vitro. Compared with baseline levels, lung BH4 concentrations decreased by 24% at 3.5 days after TBI, an effect that was reversed by GT3. At 14 and 21 days after TBI, compensatory increases in BH4 (58% and 80%, respectively) were observed. Relative to vehicle-treated controls, both GT3 and BH4 supplementation reduced postirradiation vascular peroxynitrite production at 3.5 days (by 66% and 33%, respectively), and BH4 resulted in a 68% increase in bone marrow colony-forming units. GT3 ameliorated endothelial cell apoptosis and reduced endothelial GFRP protein levels and GFRP-GTPCH binding by decreasing transcription of the GFRP gene. BH4 bioavailability is reduced in the early postradiation phase. Exogenous administration of BH4 reduces postirradiation vascular oxidative stress. GT3 potently reduces the expression of GFRP, one of the key regulatory proteins in the BH4 pathway, and may thus exert some of its beneficial effects on postradiation free radical production partly by counteracting the decrease in BH4.

Brief Report: Interferon-γ Induces Expansion of Lin−Sca-1+C-Kit+ Cells

The balance between Th1 and Th2 cells is critical for homeostasis of the immune system. Th1 cells can also regulate hematopoietic progenitor cell homeostasis by production of oncostatin M. Here we show that Th1 cell products, but not those of Th2 cells, caused a rapid expansion of lineage(-)Sca-1(+)C-kit(+) (LSK) cells in vivo and in vitro. Among Th1 cytokines, interferon-gamma (IFNgamma) was found to play a major role in this expansion by activating the expression of Sca-1 in lineage(-)Sca-1(-)C-kit(+) cells. This process was dependent on IFNgammaR1 signaling and the STAT1 pathway. Furthermore, those IFNgamma-induced LSK cells had a higher proliferation potential than control LSK cells. In addition, while the overall production of colony-forming units in bone marrow was decreased after IFNgamma treatment, the sorted LSK cells could give rise to a higher yield of colony-forming units. Finally, the IFNgamma-induced hematopoiesis was biased toward the differentiation of myeloid lineages. Therefore, our findings demonstrated a novel role of IFNgamma in activating hematopoietic progenitor cells and provide a new insight into the clinical application of interferon.

Hemangiopoietin supports animal survival and accelerates hematopoietic recovery of chemotherapy‐suppressed mice

To determine if recombinant human hemangiopoietin (HAPO), a novel growth factor for primitive cells of hematopoietic and endothelial cell lineages, accelerates hematopoietic reconstitution after high-dose chemotherapy in vivo in mice. Male Balb/c mice after treatment of 5-fluorouracil were subcutaneously injected with HAPO or its dilution for consecutive 10 d. Their survival and body weight together with peripheral blood were routinely tested. At day 7 and 14, the numbers of bone marrow (BM) cells as well as colony-forming units (CFU) after in vitro colony culture were counted. The peripheral blood CFU and the percentage of CD34+ CD117+ cells in BM were analyzed. Transwell chamber was used for cell migratory assay. HAPO at different doses significantly increased the survival rate and body weight, with an optimal effect in the HAPO 10 microg/d group. The number of BM cells and the percentage of CD34+ CD117+ cells were also increased after HAPO administration. The number of granulocyte/macrophage CFU and granulocyte, erythroid, macrophage and megakaryocyte CFU in BM after HAPO treatment was greater than that from the HAPO dilution group. More circulating CFU could be observed after injection of HAPO. In addition, this novel cytokine had a chemotactic effect on the hematopoietic stem/progenitor cells. HAPO improves animal survival and accelerates hematopoietic reconstitution of mice after high-dose chemotherapy.

Diet-induced iron deficiency anemia and pregnancy outcome in rhesus monkeys

Iron deficiency anemia (IDA) is relatively common in the third trimester of pregnancy, but causal associations with low birth weight and compromised neonatal iron status are difficult to establish in human populations. The objective was to determine the effects of diet-induced IDA on intrauterine growth and neonatal iron status in an appropriate animal model for third-trimester IDA in women. Hematologic and iron-status measures, pregnancy outcomes, and fetal and neonatal evaluations were compared between pregnant rhesus monkeys (n = 14) fed a diet containing 10 microg Fe/g diet from the time of pregnancy detection (gestation days 28-30) and controls (n = 24) fed 100 microg Fe/g diet. By the third trimester, 79% of the iron-deprived dams and 29% of the control monkeys had a hemoglobin concentration <11 g/dL. There were also significant group differences in hematocrit, mean corpuscular volume, transferrin saturation, serum ferritin, and serum iron. At birth, the newborns of monkeys iron-deprived during pregnancy had significantly lower hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin values and a lower ratio of erythroid to total colony-forming units in bone marrow than did the control newborns. Pregnancy weight gain did not differ significantly between the iron-deprived and control dams, and the fetuses and newborns of the iron-deprived dams were not growth retarded relative to the controls. Gestation length, the number of stillbirths, and neonatal neurobehavioral test scores did not differ significantly by diet group. These data indicate that an inadequate intake of iron from the diet during pregnancy in rhesus monkeys can lead to compromised hematologic status of the neonate without indications of growth retardation or impaired neurologic function at birth.

The suppression of radiation-induced NF-κB activity by dexamethasone correlates with increased cell death in vivo

In this study, we show that dexamethasone treatment increases ionizing radiation-induced cell death by inducing the inhibitory κBα (IκBα) pathway in mice. The effect of dexamethasone on radiation-induced cell death was assessed by changes in total spleen cellularity and bone marrow colony-forming unit–granulocyte–macrophage (CFU–GM) contents after total body irradiation. While in vivo treatment of mice with dexamethasone alone (1 mg/kg/day, for 2 days) failed to elicit cell death in spleen cells, the combined treatment with dexamethasone (1 mg/kg/day, for 2 days) and γ-rays (1 or 5 Gy) caused a 50–80% reduction in total cellularity in spleen and CFU–GM contents in bone marrow. These results demonstrate that dexamethasone has a synergistic effect on radiation-induced cellular damages in vivo. Immunoblot analysis showed that dexamethasone treatment significantly increases IκBα expression in the spleens of irradiated mice. In addition, the dexamethasone treatment significantly reduced radiation-induced nuclear translocation of the nucleus factor-κB in the spleens of irradiated mice. These results indicate that dexamethasone treatment in vivo may increase radiation-induced cell damages by increasing IκBα expression in hematopoietic organs such as spleen and bone marrow.

Aryl hydrocarbon receptor mediates benzene-induced hematotoxicity.

Benzene can induce hematotoxicity and leukemia in humans and mice. Since a review of the literature shows that the CYP2E1 knockout mouse is not known to possess any benzene toxicity, the metabolism of benzene by CYP2E1 in the liver is regarded to be prerequisite for its cytotoxicity and genotoxicity, although the mechanism is not fully understood yet. Because it was found some years ago that benzene was also a substrate for CYP1A1, we investigated the involvement of the aryl hydrocarbon receptor (AhR) in benzene hematotoxicity using AhR wild-type (AhR(+/+)), heterozygous (AhR(+/-)), and homozygous (AhR(-/-)) male mice. Interestingly, following a 2-week inhalation of 300 ppm benzene (a potent dose for leukemogenicity), no hematotoxicity was induced in AhR(-/-) mice. Further, there were no changes in cellularity of peripheral blood and bone marrow (BM), nor in levels of granulocyte-macrophage colony-forming units in BM. This lack of hematotoxicity was associated with the lack of p21 overexpression, which was regularly seen in the wild-type mice following benzene inhalation. Combined treatment with two major benzene metabolites, phenol and hydroquinone, induced hemopoietic toxicity, although it was not known whether this happened due to a surprising lack of expression of CYP2E1 by AhR knockout, or due to a lack of other AhR-mediated CYP enzymes, including 1A1 (i.e., a possible alternative pathway of benzene metabolism). The former possibility, evaluated in the present study, failed to show a significant relationship between AhR and the expression of CYP2E1. Furthermore, a subsequent evaluation of AhR expression after benzene inhalation tended to show higher but less significant expression in the liver, and none in the BM, compared with sham control. Although this study failed to identify the more likely of the above-mentioned two possibilities, the study using AhR knockout mice on benzene inhalation presents the unique possibility that the benzene toxicity may be regulated by AhR signaling.

Synergistic Bone Marrow Toxicity of Pyrimethamine and Zidovudine in Murine in Vivo and in Vitro Models: Mechanism of Toxicity

Pyrimethamine (Pyr) is commonly used for treatment of toxoplasmic encephalitis in AIDS patients; however, in two clinical studies, an increased number of deaths were observed when Pyr was coadministered with zidovudine (ZDV). The BALB/c mouse was chosen as a model to study the mechanism underlying the unexpected toxicity from coadministration of these drugs. Daily administration by oral gavage of 60 mg/kg Pyr and 240 mg/kg ZDV resulted in 100% lethality after 30 days. These dose levels produced no effect when the drugs were given individually for the same period. Administration of combinations of Pyr and ZDV resulted in macrocytic anemia and leukopenia with synergistic decreases in lymphocyte and neutrophil numbers. To examine the mechanism of this hematotoxicity at the cellular level, mouse bone marrow colony-forming unit (mCFU) assays were employed. A combination of ZDV with various concentrations of Pyr resulted in synergistic decreases in numbers of erythroid and granulocyte-macrophage precursors (mCFU–E and mCFU–GM). mCFU–GM precursors appeared more sensitive than erythroid precursors to combinations of Pyr and ZDV. Incorporation of 14C-ZDV into cellular DNA was increased in a dose-dependent manner in the presence of increasing concentrations of Pyr in the mCFU–GM assay. This suggested that inhibition of dihydrofolate reductase by Pyr and accompanying inhibition of dTTP synthesis allows preferential incorporation of ZDV into DNA, with resulting strand breakage and cell death. 14C-ZDV incorporation was also observed when human GM cultures were analyzed, however, incorporation was less and required higher concentrations of Pyr.

Bone marrow colony-forming unit assay in cats with naturally occurring myelodysplastic syndromes.

Feline myelodysplastic syndromes (MDS) has been diagnosed in many cats infected with feline leukemia virus, although the pathogenesis of this hematopoietic deficiency has been unclear. In this study, we assayed the bone marrow erythroid colony-forming units (CFU-E) and granulocyte-machrophage CFUs (CFU-GM) to investigate the pathogenesis of feline MDS. The number of CFU-E colonies was decreased in 4 of 7 cats with MDS, and the number of CFU-GM colonies was also decreased in 4 cats. Furthermore, small colonies of CFU-GM were found in all 7 cases. These findings indicated that refractory cytopenia of feline MDS could be caused by abnormal maturation and differentiation of hematopoietic stem cells in bone marrow, as it is in human MDS. The pathogenesis of feline MDS might be similar to that of human MDS.