Expansion of functional human hematopoietic stem cells (HSCs) has the potential to significantly improve patient outcomes in HSC transplantation and increase the dose of HSCs in gene therapy. Though several approaches have been reported to increase HSC number, a direct comparison of the various methods to expand transplantable HSCs has not been published and clinical outcome data for the various methods remains incomplete. Here, we compared several small molecule approaches reported to expand human HSCs including HDAC inhibitors (BG45, CAY10398, CAY10433, CAY10603, Entinostat, HC Toxin, LMK235, PCI-34051, Pyroxamide, Romidepsin, SAHA, Scriptaid, TMP269, Trichostatin A, or Valproic Acid), the aryl hydrocarbon antagonist, SR1, and UM171, a small molecule with unknown mechanism, for the ability to expand phenotypic HSC during in vitro culture and to increase the number of NOD-SCID engrafting cells. Following 7 days of expansion (10-point dose-response from 10 µM to .5 nM), we found that SR1 (5-fold), UM171 (4-fold), or HDAC inhibitors (>3-35-fold) resulted in an increase in the frequency and number of phenotypic CD34+CD90+CD45RA- HSC relative to cells cultured with cytokines alone. Only SR1 (18-fold) and UM171 (8-fold), however, led to enhanced engraftment in NSG mice relative to uncultured or vehicle-cultured cells. The increase in CD34+CD90+CD45RA- cells observed during in vitro culture suggest that these compounds may be generating a false phenotype by upregulating CD90 and down-regulating CD45RA on progenitors that were originally CD34+CD90-CD45RA+. To test this hypothesis, we sorted CD34+CD90-CD45RA+ cells and cultured them in the presence of the various compounds. We found that HDAC inhibitors (33-100 fold) and UM171 (28-fold) led to upregulation of CD90 on CD34+CD90-CD45RA+ cells after 4 days in culture. Since approximately 90% of the starting CD34+ cells were CD90-, these data suggest that most of the CD34+CD90+CD45RA- cells in cultures with HDAC inhibitors and UM171 arise from upregulation of CD90 rather than expansion of true CD34+CD90+CD45RA- HSCs. Further, we observed that cells derived from CD34+CD90-CD45RA+ cells are non-functional in a colony formation assay. Functionally, we have shown that AHR antagonism is optimal for expanding the number of functional human HSCs capable of engrafting NSG mice. Mechanistically, we demonstrated that culture with HDAC inhibitors and UM171 lead to upregulation of phenotypic HSC markers on downstream progenitors. This may explain the discrepancy between robust in vitro phenotypic expansion and insufficient in vivo functional activity and suggests that HDAC inhibitors and UM171 have limited ability to expand true HSCs. These data encourage caution when interpreting in vitro expansion phenotypes without confirmatory functional transplantation data, especially as these expansion approaches move into clinical trials.
The pubertal transition of gonadotropin secretion in pigs is metabolically gated. Kisspeptin (KISS1) and neurokinin B (NKB) are coexpressed in neurons within the arcuate nucleus of the hypothalamus (ARC) and are thought to play an important role in the integration of nutrition and metabolic state with the reproductive neuroendocrine axis. The hypothesis that circulating concentrations of luteinizing hormone (LH) and expression of KISS1 and tachykinin 3(TAC3, encodes NKB) in the ARC of female pigs are reduced with negative energy balance was tested using ovariectomized, prepubertal gilts fed to either gain or lose body weight. Restricted feeding of ovariectomized gilts caused a rapid and sustained metabolic response characterized by reduced concentrations of plasma urea nitrogen, insulin, leptin, and insulin-like growth factor-1 and elevated concentrations of free fatty acids. The secretory pattern of LH shifted from one of low amplitude to one of high amplitude, which caused overall circulating concentrations of LH to be greater in restricted gilts. Nutrient-restricted gilts had greater expression of follicle-stimulating hormone and gonadotropin-releasing hormone receptor, but not LH in the anterior pituitary gland. Expression of KISS1 in the ARC was not affected by dietary treatment, but expression of TAC3 was greater in restricted gilts. These data are consistent with the idea that hypothalamic expression of KISS1 is correlated with the number of LH pulse in pig, and further indicate that amplitude of LH pulses may be regulated by NKB in the gilt.
Progesterone membrane receptor component 1 (Pgrmc1) is a cytochrome b5-related protein with wide-ranging functions studied most extensively in non-neural tissues. We previously demonstrated that Pgrmc1 is widely distributed in the brain with highest expression in the limbic system. To determine Pgrmc1 functions in cells of these regions, we compared transcriptomes of control siRNA-treated and Pgrmc1 siRNA-treated N42 hypothalamic cells using whole genome microarrays. Our bioinformatics analyses suggested that Pgrmc1 plays a role in immune functions and likely regulates proinflammatory cytokine signaling. In follow-up studies, we showed that one of these cytokines, TNFα, increased expression of rtp4, ifit3 and gbp4, genes found on microarrays to be among the most highly upregulated by Pgrmc1 depletion. Moreover, either Pgrmc1 depletion or treatment with the Pgrmc1 antagonist, AG-205, increased both basal and TNFα-induced expression of these genes in N42 cells. TNFα had no effect on levels of Rtp4, Ifit3 or Gbp4 mRNAs in mHippoE-18 hippocampal control cells, but Pgrmc1 knock-down dramatically increased basal and TNFα-stimulated expression of these genes. P4 had no effect on gbp4, ifit3 or rtp4 expression or on the ability of Pgrmc1 to inhibit TNFα induction of these genes. However, a majority of the top upstream regulators of Pgrmc1 target genes were related to synthesis or activity of steroids, including P4, that exert neuroprotective effects. In addition, one of the identified Pgrmc1 targets was Nr4a1, an orphan receptor important for the synthesis of most steroidogenic molecules. Our findings indicate that Pgrmc1 may exert neuroprotective effects by suppressing TNFα-induced neuroinflammation and by regulating neurosteroid synthesis.
Bone Marrow Transplant (BMT) is a potentially curative treatment for malignant and non-malignant blood disorders. Current regimens for patient preparation, or conditioning, prior to BMT limit the use of this curative procedure due to regimen-related mortality and morbidities, including risks of organ toxicity, infertility and secondary malignancies. Targeted preparation using antibody drug conjugates (ADCs) to mouse CD45 has previously been shown to be sufficient to enable bone marrow transplant (BMT) in syngeneic immune competent mice (Palchaudhuri et al. Nature Biotech 2016 34:738–745), and this approach to preparation has the potential to expand the utility of BMT if it can be successfully translated to patients. To further investigate the utility of this tool ADC in murine transplant models, we explored anti-CD45-saporin (CD45-SAP) in an allogeneic minor mismatch transplant model (Balb/c donor into DBA/2 recipients). The goal of the work was to identify the level of immune suppression, if any, that needs to be used in combination with CD45-SAP to enable high donor chimerism in the allogeneic setting.CD45-SAP (1.9 mg/kg, iv) was evaluated alone or in combination with additional immune modulating agents: clone 30F11 (25 mg/kg, IP), a naked anti-CD45 antibody that mimics ATG by relying on effector function to enable potent peripheral B- and T -cell depletion; pre-transplant Cytoxan (PreTCy, 200 mg/kg, IP), 2 Gy total body irradiation (TBI), and post-transplant Cytoxan (PTCy, 200 mg/kg, IP) to prevent graft versus host disease as well as block host versus graft rejection. 9 Gy TBI was used as the conventional conditioning positive control. Conditioned mice were transplanted with 2 × 107 whole bone marrow cells, and chimerism assessed over 12 weeks.CD45-SAP in combination with PTCy achieved comparable peripheral myeloid chimerism, a readout of stem cell engraftment, to 9 Gy TBI (>90%) at 8 weeks post-transplantation (Fig 1). The addition of 30F11 to the CD45-SAP/PTCy protocol had no effect on peripheral donor chimerism (59% vs 61%), suggesting additional lymphodepletion is not required. In contrast, the single agents alone, 2 Gy TBI in combination with 30F11 and PTCy resulted in <5% donor engraftment. Other conditions tested that achieved low level donor myeloid chimerism were multi-dosing of 30F11 (QDx3) plus 2 Gy TBI with PTCy (40% donor chimerism) and PreTCy plus 30F11 (QDx3) with PTCy (20% donor chimerism). For all groups, stem cell chimerism in the bone marrow matched the peripheral chimerism.These results indicate CD45-SAP in combination with PTCy is sufficient to enable high levels of donor chimerism in the minor mismatched setting without the need for additional immune suppression. CD45-SAP was more effective at conditioning than 2Gy TBI or PreTCy. Future experiments will investigate the CD45-SAP/PTCy protocol in haploidentical and fully mismatched allogeneic mouse models. Bone Marrow Transplant (BMT) is a potentially curative treatment for malignant and non-malignant blood disorders. Current regimens for patient preparation, or conditioning, prior to BMT limit the use of this curative procedure due to regimen-related mortality and morbidities, including risks of organ toxicity, infertility and secondary malignancies. Targeted preparation using antibody drug conjugates (ADCs) to mouse CD45 has previously been shown to be sufficient to enable bone marrow transplant (BMT) in syngeneic immune competent mice (Palchaudhuri et al. Nature Biotech 2016 34:738–745), and this approach to preparation has the potential to expand the utility of BMT if it can be successfully translated to patients. To further investigate the utility of this tool ADC in murine transplant models, we explored anti-CD45-saporin (CD45-SAP) in an allogeneic minor mismatch transplant model (Balb/c donor into DBA/2 recipients). The goal of the work was to identify the level of immune suppression, if any, that needs to be used in combination with CD45-SAP to enable high donor chimerism in the allogeneic setting. CD45-SAP (1.9 mg/kg, iv) was evaluated alone or in combination with additional immune modulating agents: clone 30F11 (25 mg/kg, IP), a naked anti-CD45 antibody that mimics ATG by relying on effector function to enable potent peripheral B- and T -cell depletion; pre-transplant Cytoxan (PreTCy, 200 mg/kg, IP), 2 Gy total body irradiation (TBI), and post-transplant Cytoxan (PTCy, 200 mg/kg, IP) to prevent graft versus host disease as well as block host versus graft rejection. 9 Gy TBI was used as the conventional conditioning positive control. Conditioned mice were transplanted with 2 × 107 whole bone marrow cells, and chimerism assessed over 12 weeks. CD45-SAP in combination with PTCy achieved comparable peripheral myeloid chimerism, a readout of stem cell engraftment, to 9 Gy TBI (>90%) at 8 weeks post-transplantation (Fig 1). The addition of 30F11 to the CD45-SAP/PTCy protocol had no effect on peripheral donor chimerism (59% vs 61%), suggesting additional lymphodepletion is not required. In contrast, the single agents alone, 2 Gy TBI in combination with 30F11 and PTCy resulted in <5% donor engraftment. Other conditions tested that achieved low level donor myeloid chimerism were multi-dosing of 30F11 (QDx3) plus 2 Gy TBI with PTCy (40% donor chimerism) and PreTCy plus 30F11 (QDx3) with PTCy (20% donor chimerism). For all groups, stem cell chimerism in the bone marrow matched the peripheral chimerism. These results indicate CD45-SAP in combination with PTCy is sufficient to enable high levels of donor chimerism in the minor mismatched setting without the need for additional immune suppression. CD45-SAP was more effective at conditioning than 2Gy TBI or PreTCy. Future experiments will investigate the CD45-SAP/PTCy protocol in haploidentical and fully mismatched allogeneic mouse models.
Allogeneic bone marrow transplant (BMT) is a potentially curative approach in patients with refractory or high-risk hematologic malignancies, like acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Current regimens for patient preparation, or conditioning, prior to BMT limit the use of this procedure due to regimen-related mortality and morbidities. As a result, many eligible patients do not consider transplant and only 2/3 those transplanted are able to tolerate a reduced intensity conditioning regimen, which is associated with increased relapse (Scott, J Clin Onc 2017). Thus, safer and more effective conditioning agents with improved disease control are urgently needed. We developed two novel antibody drug conjugates (ADCs) conjugated to amanitin (AM) targeting CD117 (C-KIT, Pearse 2018), which is expressed on hematopoietic stem, progenitor, AML and MDS cells in >60% of patients (Ludwig, Haematologica 1997). CD45 (Palchaudhuri 2018) is expressed on all lympho-hematopoietic cells and hematologic malignancies except multiple myeloma. We aim to design ADC-based conditioning agents with the dual benefit of specifically depleting primary human hematopoietic stem progenitor cells (HSPCs) to enable BMT while reducing disease burden in leukemia models.ADCs were tested in xenograft murine models inoculated with human AML cells from immortalized cell lines (Kasumi-1, CD117 expressing leukemia; REH-Luc, CD45 expressing AML), and three AML patient-derived xenografts (PDX) expressing both CD117 and CD45 (2 of 3 are from patients that relapsed post allogeneic BMT).In the Kasumi model, a single dose of 0.3 mg/kg anti-CD117-AM on day 7 or 42 after inoculation markedly increased survival (median >240 days) compared to vehicle (median 76 days) or unconjugated anti-CD117 antibody (median 86.5 days) (n=6-8/group, p<0.01). In the REH-Luc model, a single dose of 1 mg/kg anti-CD45-AM on day 5 after inoculation increased median survival by 15 days compared to vehicle or unconjugated anti-CD45 antibody (n=10/group, p<0.01). In the PDX models, a single dose of ADCs (anti-CD117-AM, anti-CD45-AM, isotype-AM (ISO-AM), unconjugated anti-CD117 antibody, or vehicle PBS) was administered when 2-5% blasts were observed in the blood (n=4-5/group). Survival was significantly increased with 1 mg/kg anti-CD117-AM and 1 mg/kg anti-CD45-AM as compared to vehicle (Figure 1 and Table 1).Anti-CD117-AM and anti-CD45-AM are potent anti-leukemia agents based on these data in humanized murine models with established AML. Together with prior reports on the potency of anti-CD117-AM and anti-CD45-AM as conditioning agents, these non-genotoxic ADCs may be useful in reducing disease burden in patients and in recipients of reduced-dose conditioning who are at high risk of disease relapse. Allogeneic bone marrow transplant (BMT) is a potentially curative approach in patients with refractory or high-risk hematologic malignancies, like acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Current regimens for patient preparation, or conditioning, prior to BMT limit the use of this procedure due to regimen-related mortality and morbidities. As a result, many eligible patients do not consider transplant and only 2/3 those transplanted are able to tolerate a reduced intensity conditioning regimen, which is associated with increased relapse (Scott, J Clin Onc 2017). Thus, safer and more effective conditioning agents with improved disease control are urgently needed. We developed two novel antibody drug conjugates (ADCs) conjugated to amanitin (AM) targeting CD117 (C-KIT, Pearse 2018), which is expressed on hematopoietic stem, progenitor, AML and MDS cells in >60% of patients (Ludwig, Haematologica 1997). CD45 (Palchaudhuri 2018) is expressed on all lympho-hematopoietic cells and hematologic malignancies except multiple myeloma. We aim to design ADC-based conditioning agents with the dual benefit of specifically depleting primary human hematopoietic stem progenitor cells (HSPCs) to enable BMT while reducing disease burden in leukemia models. ADCs were tested in xenograft murine models inoculated with human AML cells from immortalized cell lines (Kasumi-1, CD117 expressing leukemia; REH-Luc, CD45 expressing AML), and three AML patient-derived xenografts (PDX) expressing both CD117 and CD45 (2 of 3 are from patients that relapsed post allogeneic BMT). In the Kasumi model, a single dose of 0.3 mg/kg anti-CD117-AM on day 7 or 42 after inoculation markedly increased survival (median >240 days) compared to vehicle (median 76 days) or unconjugated anti-CD117 antibody (median 86.5 days) (n=6-8/group, p<0.01). In the REH-Luc model, a single dose of 1 mg/kg anti-CD45-AM on day 5 after inoculation increased median survival by 15 days compared to vehicle or unconjugated anti-CD45 antibody (n=10/group, p<0.01). In the PDX models, a single dose of ADCs (anti-CD117-AM, anti-CD45-AM, isotype-AM (ISO-AM), unconjugated anti-CD117 antibody, or vehicle PBS) was administered when 2-5% blasts were observed in the blood (n=4-5/group). Survival was significantly increased with 1 mg/kg anti-CD117-AM and 1 mg/kg anti-CD45-AM as compared to vehicle (Figure 1 and Table 1). Anti-CD117-AM and anti-CD45-AM are potent anti-leukemia agents based on these data in humanized murine models with established AML. Together with prior reports on the potency of anti-CD117-AM and anti-CD45-AM as conditioning agents, these non-genotoxic ADCs may be useful in reducing disease burden in patients and in recipients of reduced-dose conditioning who are at high risk of disease relapse. Figure 1 and Table 1Table 1Table of PDX AML models' median survival (days post dose administration) and statistical analysis (Log-Rank Test) comparing ADCs against either control group (PBS, ISO-AM, and/or unconjugated antibody). Open table in a new tab
