Bone Marrow Recipients Research Articles

IL1RAP-specific T cell engager depletes acute myeloid leukemia stem cells

BackgroundThe interleukin-1 receptor accessory protein (IL1RAP) is highly expressed on acute myeloid leukemia (AML) bulk blasts and leukemic stem cells (LSCs), but not on normal hematopoietic stem cells (HSCs), providing an opportunity to target and eliminate the disease, while sparing normal hematopoiesis. Herein, we report the activity of BIF002, a novel anti-IL1RAP/CD3 T cell engager (TCE) in AML.MethodsAntibodies to IL1RAP were isolated from CD138+ B cells collected from the immunized mice by optoelectric positioning and single cell sequencing. Individual mouse monoclonal antibodies (mAbs) were produced and characterized, from which we generated BIF002, an anti-human IL1RAP/CD3 TCE using Fab arm exchange. Mutations in human IgG1 Fc were introduced to reduce FcγR binding. The antileukemic activity of BIF002 was characterized in vitro and in vivo using multiple cell lines and patient derived AML samples.ResultsIL1RAP was found to be highly expressed on most human AML cell lines and primary blasts, including CD34+ LSC-enriched subpopulation from patients with both de novo and relapsed/refractory (R/R) leukemia, but not on normal HSCs. In co-culture of T cells from healthy donors and IL1RAPhigh AML cell lines and primary blasts, BIF002 induced dose- and effector-to-target (E:T) ratio-dependent T cell activation and leukemic cell lysis at subnanomolar concentrations. BIF002 administered intravenously along with human T cells led to depletion of leukemic cells, and significantly prolonged survival of IL1RAPhigh MOLM13 or AML patient-derived xenografts with no off-target side effects, compared to controls. Of note, BiF002 effectively redirects T cells to eliminate LSCs, as evidenced by the absence of disease initiation in secondary recipients of bone marrow (BM) from BIF002+T cells-treated donors (median survival not reached; all survived > 200 days) compared with recipients of BM from vehicle- (median survival: 26 days; p = 0.0004) or isotype control antibody+T cells-treated donors (26 days; p = 0.0002).ConclusionsThe novel anti-IL1RAP/CD3 TCE, BIF002, eradicates LSCs and significantly prolongs survival of AML xenografts, representing a promising, novel treatment for AML.

Abstract 2052: Macrophage-specific Ripk1 Knockdown Reduces Macrophage Inflammation And Atherosclerosis Progression In Ldlr -/- Mice.

Background: Chronic activation of the innate immune system drives inflammation and contributes to atherosclerosis. Pro-inflammatory agents (oxidised LDL, IL-1β) drive lesional inflammation and development, whereas anti-inflammatory agents (IL-10, HDL, Apolipoprotein-AI), promote an anti-inflammatory milieu and resolution. RIPK1 (Receptor interacting protein kinase 1) is a master regulatory gene that decides whether a cell undergoes inflammation, apoptosis and/or necroptosis, depending on its phosphorylation or ubiquitination status. We have previously observed that RIPK1 gene expression is increased in both endothelial cells and macrophages in early atherosclerotic lesions (Karunakaran et al, Circulation 2022). Here, we seek to determine whether macrophage-specific RIPK1 contributes to atherosclerosis. Methods: Given that Ripk1 -/- mice are lethal, we performed bone marrow (BM) transplants of Ripk1 +/- and wild-type (wt) littermate BM into female and male Ldlr -/- mice (n=8-12/grp) and mice were fed a western diet for 12-16 wks. In vitro experiments were performed in BM derived macrophages from wt and Ripk1 +/- mice. Results: Ldlr -/- mice recipient of Ripk1 +/- BM and western diet feeding for 12 or 16 wk had a marked reduction in aortic sinus lesion area (68.9% and 49.7% respectively, p<0.05) compared to their wt controls. Preliminary analysis show that there is a reduction in lesional macrophage area in Ripk1 +/- BM recipient Ldlr -/- mice relative to control (0.02±0.01 vs 0.01±0.006mm, p<0.01), suggesting that macrophage infiltration was reduced. Interestingly, anti-inflammatory agents, including recombinant IL10, IL13 (both 100ng/ml), Apolipoprotein-AI and discoidal HDL (both 500ug/mL) markedly reduced macrophage Ripk1 expression in vitro (by ~50%, p<0.05), likely reducing Ripk1-dependent inflammation and promoting an anti-inflammatory milieu. Current mechanistic studies are investigating the transcriptional regulator, STAT3, as a key regulator of Ripk1 expression. Conclusion: We identified macrophage-RIPK1 as a central driver of atherosclerosis. These timely studies highlight the therapeutic potential of dampening Ripk1 overexpression, as opposed to the detrimental abolishment of Ripk1, in treating atherosclerosis.

Complications of total-body irradiation in allogeneic bone marrow transplantation: A review article

Hematopoietic stem cell transplantation commonly known as bone marrow transplantation (BMT) or allogeneic BMT (using healthy blood stem cells from a donor) is the preferred therapeutic option for numerous blood-related conditions, both malignant and non-malignant. It is often the sole therapy strategy and essential for relapsed and refractory hematologic malignancies. There have studies regarding BMT on regimen containing total body irradiation (TBI) and a regimen without TBI. It is expected that TBI-based conditioning regimens provide better antitumor effects than chemotherapy regimens. The primary objective of TBI is to eradicate the recipient's bone marrow, facilitating the successful engraftment of donor bone marrow. Acute lymphoid leukemia (ALL) is the principal indication for TBI in bone marrow transplantation. Other diseases including Hodgkin’s lymphoma, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), multiple myeloma (MM), etc., may benefit from TBI-based regimens; however, TBI use is associated with many side effects.The main complications of patients who underwent TBI-containing conditioning regimens in bone marrow transplantation are vomiting and nausea, with frequencies of approximately 66% and 35%, respectively. However, these events are easily managed. Acute complications include stomatitis, diarrhea, loss of appetite, temporary loss of taste, rash and asthenia. Moreover, veno-occlusive disease, interstitial pneumonitis, lung side effects, growth hormone deficiency, neurological side effects, cataracts, renal toxicity, endocrine impairments, and infertility are other complications in patients who underwent TBI-containing conditioning regimens in bone marrow transplantation. In review article, a complication of total-body irradiation in allogeneic bone marrow transplantation was assessed.

Alloengraftment without significant toxicity or GVHD in CD45 antibody-drug conjugate–conditioned Fanconi anemia mice

AbstractFanconi anemia (FA) is an inherited DNA repair disorder characterized by bone marrow (BM) failure, developmental abnormalities, myelodysplasia, leukemia, and solid tumor predisposition. Allogeneic hematopoietic stem cell transplantation (allo-HSCT), a mainstay treatment, is limited by conditioning regimen–related toxicity and graft-versus-host disease (GVHD). Antibody-drug conjugates (ADCs) targeting hematopoietic stem cells (HSCs) can open marrow niches permitting donor stem cell alloengraftment. Here, we report that single dose anti-mouse CD45–targeted ADC (CD45-ADC) facilitated stable, multilineage chimerism in 3 distinct FA mouse models representing 90% of FA complementation groups. CD45-ADC profoundly depleted host stem cell enriched Lineage−Sca1+cKit+ cells within 48 hours. Fanca−/− recipients of minor-mismatched BM and single dose CD45-ADC had peripheral blood (PB) mean donor chimerism >90%; donor HSCs alloengraftment was verified in secondary recipients. In Fancc−/− and Fancg−/− recipients of fully allogeneic grafts, PB mean donor chimerism was 60% to 80% and 70% to 80%, respectively. The mean percent donor chimerism in BM and spleen mirrored PB results. CD45-ADC–conditioned mice did not have clinical toxicity. A transient <2.5-fold increase in hepatocellular enzymes and mild-to-moderate histopathological changes were seen. Under GVHD allo-HSCT conditions, wild-type and Fanca−/− recipients of CD45-ADC had markedly reduced GVHD lethality compared with lethal irradiation. Moreover, single dose anti–human CD45-ADC given to rhesus macaque nonhuman primates on days −6 or −10 was at least as myeloablative as lethal irradiation. These data suggest that CD45-ADC can potently promote donor alloengraftment and hematopoiesis without significant toxicity or severe GVHD, as seen with lethal irradiation, providing strong support for clinical trial considerations in highly vulnerable patients with FA.

Differential clinical impact of letermovir prophylaxis according to graft sources: a KSGCT multicenter retrospective analysis

AbstractClinically significant cytomegalovirus infection (csCMVi) is frequently observed after allogeneic hematopoietic stem cell transplantation (HSCT) and prophylaxis with letermovir is commonly adopted. However, the clinical benefit of letermovir prophylaxis according to graft sources has not been sufficiently elucidated. We retrospectively analyzed 2194 recipients of HSCT who were CMV-seropositive (236 with letermovir prophylaxis and 1958 without prophylaxis against CMV). csCMVi was significantly less frequent in patients with letermovir prophylaxis than in those without (23.7% vs 58.7% at 100 days after HSCT, P < .001) and the same trend was seen when recipients of bone marrow (BM), peripheral blood stem cell (PBSC), or cord blood (CB) transplantation were separately analyzed. In recipients of BM, nonrelapse mortality (NRM) was significantly lower in the letermovir group at 6 months after HSCT (5.0% vs 14.9%, P = .018), and the same trend was observed in recipients of PBSCs (14.7% vs 24.8%, P = .062); however, there was no statistical significance at 1 year (BM, 21.1% vs 30.4%, P = .67; PBSCs, 21.2% vs 30.4%, P = .096). In contrast, NRM was comparable between recipients of CB with and without letermovir prophylaxis throughout the clinical course (6 months, 23.6% vs 24.3%, P =.92; 1 year, 29.3% vs 31.0%, P = .77), which was confirmed by multivariate analyses. In conclusion, the impact of letermovir prophylaxis on NRM and csCMVi should be separately considered according to graft sources.

Bcor Truncation Mutations Collaborate with a U2AF1 S34F Mutant to Promote Clonal Hematopoiesis and MDS

Mutations in spliceosome genes such as U2AF1 have been identified across major cancer types, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Mutations involving the U2AF1 gene, mostly commonly involving amino acids 34 or 157 have been reported in up to 20% of patients with MDS. To investigate how mutant U2AF1 contribute to hematologic malignancy, we generated transgenic mice that expressed a mutant U2AF1 S34F mutant under control of hematopoietic specific Vav1 regulatory elements. Sanger sequencing demonstrated that expression of the mutant U2AF1 was similar to that of wild-type (WT) U2AF1 in bone marrow and spleen. U2AF1 S34F mice showed decreased long-term hematopoietic stem cells (HSC) compared with age matched wild type WT mice. Hematopoietic stem cell transplantation (HSCT) showed that U2AF1 S34F HSCs repopulated recipients less efficiently than WT HSCs, consistent with previous reports. However, U2AF1 S34F mice did not develop evidence of hematologic malignancy, suggesting that additional mutations were required to develop MDS or AML. BCOR, a corepressor for BCL6, has been reported to be frequently co-mutated in MDS patients with U2AF1 S34F, suggesting that these two mutations might collaborate to produce MDS. To test the hypothesis, we introduced a BCOR truncation mutation into hematopoietic stem and progenitor cells (HSPC) from U2AF1 S34F or WT mice, using CRISPR-Cas9, followed by transplant to WT recipient mice. Primary recipients of U2AF1 S34F and Bcor double mutant bone marrow (U2B) showed decreased engraftment compared to WT-Bcor mutant (WB) recipients, consistent with a competitive disadvantage of U2AF1 S34F HSPC observed previously by several labs. We performed secondary HSCT to evaluate the long-term repopulation pattern of U2B HSPC. Secondary transplant recipients from U2B primary mice showed anemia and leukopenia, consistent with MDS. U2B secondary recipients typically showed low engraftment initially (reflecting ineffective hematopoiesis) followed by gradually increasing proportion of U2B cells in the peripheral blood. At necropsy, all secondary recipients of U2B bone marrow showed evidence of clonal hematopoiesis involving cells with 1 or 2 distinct, unique Bcor truncation mutations. We then sequenced bone marrow DNA from the U2B mouse used as donor for the secondary transplant and identified 14 independent Bcor mutations. All 14 Bcor mutations were out of frame, and encoded a truncated Bcor protein, indicating that cells with a U2AF1 S34F and a Bcor truncation had a competitive advantage in vivo. These results suggest that loss of normal Bcor function can collaborate with a U2AF1 S34F mutant, resulting in clonal expansion of abnormal HSC.

The DNA Sensor AIM2 Determines B Cell Fate after BCR Activation in Chronic Graft-Vs-Host Disease

Prevention and treatment of chronic graft-vs-host disease (cGVHD), a debilitating late immune toxicity in patients after allogeneic hematopoietic stem cell transplantation (allo-HCT), who are otherwise cured of hematological malignancies, remains a major challenge. T cells incite and promote cGVHD through coordinated T-B cell responses. Alloantibody production ( Srinivasan et al, Blood 2012, Jin et al, Blood 2016) and aberrant B Cell Receptor (BCR) signaling ( Allen et al, Blood 2014, Flynn et al, Blood 2015) play fundamental roles in the pathology of cGVHD. B cell-intrinsic pathways in cGVHD remain incompletely understood, limiting our therapeutic capacity for patients. In a single cell RNA sequencing study, we found that transcript levels of the DNA sensor Absent in Melanoma 2 (AIM2) were significantly increased in CD27+ BCR-activated B cell clusters in circulating as well as in tissue-resident cGVHD B cells ( Poe et al, JCI Insight 2023). After confirming AIM2 expression is restricted to the nucleus, we also previously showed that AIM2-expressing B cells were augmented after BCR and interferon-gamma (IFNγ) and/or Toll-like receptor (TLR)-7 or 9 stimulation ( Visentin J, Blood 2022;140 Supp 1), suggesting a context-dependent role for AIM2 after allo-HCT. Our new findings now afford the hypothesis that an AIM2-BCR signaling axis determines B cell fate after allo-HCT. To address this hypothesis, we first examined whether AIM2 modulates the promotion of antibody-secreting cells (ASCs) after BCR activation using a hapten-carrier immunization model. We compared numbers of hapten-specific ASCs from immunized WT and AIM2-KO mouse splenocytes using enzyme-linked immunosorbent spot (ELISPOT). We compared numbers of ASCs on day 3 post-immunization, when early germinal center (GC) formation is known to occur. We found that hapten-specific IgM ASCs were significantly increased in WT splenocytes compared to AIM2-KO (p=0.002). By contrast, when we looked at day 8, when mature GC formation and rapid ASC proliferation is known to occur, we found that AIM2-KO immunized mice had significantly lower numbers of hapten-specific IgM (p=0.005) and IgG (p=0.004) ASCs in AIM2-expressing mice (Figure 1a). Our results suggest a role for AIM2 in regulating the survival of short-lived plasmablast-like ASCs after high affinity antigen encounter and maturation of germinal centers. To further investigate whether AIM2 affects expansion of GC-derived (GL7+CD95+) ASCs, we stimulated B cells purified from wildtype (WT) or global AIM2-deficient (AIM2-KO) mice in vitro with lipopolysaccharide (LPS) and IL-4 +/- BCR stimulation. We found that AIM2-deficient B cells, especially GC-like cell populations, expanded significantly in an anti-IgM concentration-dependent manner up to 12-fold (p=0.03) after 72 hr (Figure 1b). By contrast, B cell expansion was constrained in AIM2-sufficient B cells. This expansion was unlikely to be due to differences in proliferation alone, as we did not detect differential Ki67 expression in stimulated B cells with or without AIM2. Together, these data suggest a role for AIM2 in modulating BCR signaling. Finally, we examined the role of AIM2 in BCR-mediated cell death after chronic antigen exposure using our previously described bone marrow transplant (BMT) model, where recipients of bone marrow and splenocytes (BM+Spl) exhibit cGVHD manifestations ( Poe et al, JCI Insight 2018, Jia et al, Blood 2021). We have previously generated C57BL/6J mice with conditional deficiency of AIM2 only in B cells (AIM2 fl/flMb1 Cre/+, CKO), and used these in our BMT model as donors. At day 58 post BMT, we performed ex vivo stimulation of splenic B cells from BMT recipients. While B cells from CKO BM+Spl recipients expanded ex vivo in an anti-IgM concentration-dependent manner, B cells from AIM2-sufficient BM+Spl recipients demonstrated limited responsiveness ex vivo to BCR stimulation. This extends our in vitro observations by suggesting a role for AIM2 in B cell tolerance after chronic antigen exposure. Ongoing experiments will determine if B cell-intrinsic AIM2 contributes to cGVHD pathogenesis. Thus far, our data reveal a novel role for AIM2 as a B cell survival checkpoint after BCR activation. Further delineation of context-dependent and synergistic AIM2-BCR signaling after allo-HCT will lead to strategies that block the development of cGVHD without adversely affecting anti-tumor activity.

Dynamic Oscillation of Tetraspanin CD9 Orchestrates Engraftment and Mobilization of Human Hematopoietic Stem/Progenitor Cells

Background Trafficking of hematopoietic stem/progenitor cells (HSPCs) to and from their bone marrow (BM) niche is pivotal for engraftment and mobilization during clinical transplantation, where both processes are controlled by highly orchestrated molecular programs. We previously identified the tetraspanin CD9 as a downstream effector of the SDF-1/CXCR4 axis driving migration, adhesion and homing of human CD34+ HSPCs, but its consequential impact on stem cell engraftment and mobilization as well as the underlying mechanisms remains unknown. Methods Umbilical cord blood (UCB) CD34+ cells were exposed to a CD9 neutralizing antibody ALB6 prior to infusion into sublethally irradiated NOD/SCID mice, with overall engraftment and lineage output assessed by enumerating human CD45+ (pan-leukocytes), CD19+ (B-lymphoid), CD33+ (myeloid) and CD34+ (stem/progenitors) cells in the recipient BM at 8 weeks post-transplantation. CD9 expression on engrafted HSPCs was characterized by flow cytometry and compared to those before transplantation or after G-CSF mobilization. The oscillation pattern was then validated on HSPCs in pairwise BM, peripheral blood (PB) and G-CSF-mobilized peripheral blood (MPB) samples from healthy donors. Activity of integrin VLA-4 (an established binding partner of CD9) was evaluated on BM-residing and mobilized HSPCs by VCAM-1 binding assay. Results A short exposure of UCB CD34+ cells to the CD9 neutralizing antibody markedly inhibited their BM engraftment by 64.2% ( P=0.003), reflected by the general decline in human CD45+ cell population. There was no bias in lymphoid/myeloid lineage outputs, suggesting CD9-mediated engraftment is not restricted to specific subsets of hematopoietic precursors. CD9 expression was drastically elevated on engrafted CD34+ cells in the BM from 9.6% to 91.4% ( P&amp;lt;0.001) compared to pre-transplantation. In contrast, CD9 expression was significantly downregulated on BM CD34+ cells from 73.4% to 33.5% ( P&amp;lt;0.001) upon G-CSF mobilization, with the decline observed in both primitive stem cells (CD34+CD38-) and committed progenitors (CD34+CD38+), indicating that the oscillation of CD9 is highly associated with HSPC trafficking. Concordantly, pairwise analysis of steady-state HSPCs in BM and PB from healthy donors revealed higher CD9 level in BM-residing CD34+ cells than the PB-circulating portion (46.1% vs. 26.5%, P=0.004; n=13). In addition, a significantly higher CD9 expression was observed on BM CD34+ cells (47.7%; n=16) than those in MPB (32.3%, n=25; P=0.003). CD9 expression on MPB CD34+ cells was enhanced upon SDF-1 exposure ( P=0.006), revealing a dynamic regulation. In normal BM, CD9+ HSPCs exhibited elevated VLA-4 activities with preferential binding to soluble VCAM-1 compared to the CD9- counterpart (51.4% vs. 25.9%, P&amp;lt;0.001; n=10). Superiority in VCAM-1 binding capacity was also witnessed in BM-residing CD34+ cells compared to MPB (37.7% vs. 4.1%, P=0.014), implicating that the influence of CD9 on HSPC trafficking may due to the alteration of integrin activities and downstream adhesive properties. Conclusion This study describes the dynamic regulation of CD9 during entry and exit of HSPCs to and from their BM niche via modulation of integrin activities. The findings extend our understandings on HSPC trafficking that could potentially lead to new strategies to enhance clinical stem cell transplantation and mobilization.

Myelodysplastic Syndromes Benefit from Iron-Restricted Bone Marrow Transplant in Preclinical Mouse Model

Background: Hematopoietic stem cell transplantation (HSCT) remains the only fully curative therapy for diseases such as b-thalassemia, sickle cell disease, MDS and AML and represents a relevant procedure for gene therapy .Tissue iron overload and non-transferrin bound iron (NTBI) have emerged as adverse prognostic factors for post-transplant survival. Recently, we showed that - in line with observations in transplanted individuals - preparatory chemotherapy conditioning with a combination of busulfan and fludarabine in wild-type mice resulted in elevated serum iron and transferrin (Tf) saturation and elicited NTBI formation. Tf saturation and NTBI levels increased proportionally with conditioning intensity. Interestingly, we found that pre-conditioning TMPRSS6 silencing through a GalNAc-conjugated siRNA - by increasing hepcidin levels - reduced the peri-transplant elevation of Tf saturation and limited the appearance of NTBI. These results proved that NTBI formation is induced by conditioning chemotherapy in the peri-trasplant period independently of the presence of pre-existing iron overload. Aims &amp; Methods: Here we asked whether these observations hold true in conditions hallmarked by pre-existing iron overload and elevated NTBI and how iron restriction impacts on bone marrow transplant (BMT) outcome. To this aim, we applied the GalNAc TMPRSS6 siRNA as iron restriction strategy in a mouse model of myelodysplastic syndromes (MDS) and assessed its ability to reduce conditioning-elicited NTBI formation as well as its potential benefit on BMT. After TMPRSS6 siRNA administration, CD45.2 + MDS mice received conditioning chemotherapy and transplant of CD45.1 + wild-type bone marrow cells. Systemic iron parameters, engraftment and chimerism were then analyzed. Results: Importantly, conditioning exacerbated the elevated systemic iron levels in MDS mice, which was prevented by pre-conditioning TMPRSS6 silencing. Similar to our observation in wild-type mice, recipient CD45.2 + MDS mice showed a superior blood chimerism, resulting from increased bone marrow and spleen engraftment of CD45.1 + donor cells. Multilineage engraftment in bone marrow, spleen and peripheral blood was superior in iron-restricted recipient MDS mice compared to controls. Finally, donor chimerism of hematopoietic stem and progenitor cells (HSPCs) was higher in iron-restricted versus control MDS mice undergoing BMT. Conclusion: These findings demonstrate that pre-transplant conditioning profoundly alters iron homeostasis and restricting iron improves BMT outcome by increasing engraftment efficiency. Overall, these data suggest that conditioning-elicited NTBI is a critical mediator of peri-transplant toxicity and adversely impacts transplant outcome. Specifically, our observations prove that iron restriction improves donor HSPC engraftment in the recipient bone marrow, likely resulting in more efficient reconstitution of the HSPC pool and improved multilineage engraftment. Lastly, our results indicate that the peri-transplant application of novel iron restriction strategies aimed at reducing conditioning-induced NTBI - by positively affecting BMT outcome - is of potential benefit for patients undergoing BMT or gene therapy, presenting either with or without pre-transplant iron overload.

Septin11 and Septin 8 Play Critical and Overlapping Roles Murine Stem Cell Function

Septins are highly conserved GTP binding proteins which regulate a variety of cellular functions including cytokinesis, polarity, cell cycle, vesicle trafficking, and exocytosis and are now considered the fourth component of the cytoskeleton. Septins are implicated in T and B cell lymphomas; and acute myeloid leukemias, where they frequently act as fusion partners with the Mixed-Lineage Leukemia (MLL) gene. Previous data in our laboratory led to the discovery of a small molecular inhibitor, DW0254, which inhibits RAS function and its downstream target RhoGTPase, Rac, including the hematopoietic-specific Rac2 GTPase, also implicated in MLL leukemias. The inhibitor leads to apoptosis of leukemia cell lines. Using biophysical methods, we discovered that DW0254 binds both the RAS chaperone, PDE6D, and Septin11, which is a member of the Septin6 family (Sara CN, et al., Blood Cancer 2022). Although Septins have been reported to be involved in function of the RhoGTPase Cdc42 and hematopoietic stem progenitor cell (HSPC) polarity, the role of Septin11 in hematopoiesis has not been reported. Septin11 is a member of the Septin6 family of proteins which consists of Septin6, 8, 10, 11 and 14 with significant homology between family members. Previous data in yeast has hypothesized functional overlap within each family of Septin proteins. To better understand the functional role of Septin11, we created lentivirus vectors tagged with a GFP reporter that contained shmiRs against Septin11. Culture of Septin11 knockdown (Septin11 KD) murine HSPC in IMDM (10ng/ml IL-3, 25ng/ml of SCF, TPO, Flt-3 and IL-6) resulted in an average of 13-fold decrease in cell counts compared to controls (p=0.02) and a decrease in total clonogenic activity (Figure 1) with defects seen in both myeloid (GM) and erythroid (BFU-E) colonies. Reduced clonogenic numbers were accompanied by a significant increase in early apoptotic cells suggesting that Septin11 is important in regulating apoptosis in HSPC. To determine the potential role of Septin11 in more primitive hematopoietic cells, we utilized competitive repopulation assays. Transduced BoyJ HSPC (GFP+ Septin11 KD and BFP+ control, mixed 1:1) were injected into lethally irradiated C57Bl/6 mice. We observed a reduction in multilineage chimerism in the peripheral blood, bone marrow, spleen and thymus in primary transplant recipients after knockdown of Septin11 (Table 1). To better define function in specific hematopoietic populations of HSPC, we performed knockdown in FACS-sorted purified cells. Engraftment studies demonstrated a significant reduction in bone marrow chimerism in primary transplant recipients after knockdown of Septin11 most clearly in LT-HSC and ST-HSC (Table 1) with no statistically significant change in MPP. Therefore, to better study the hematopoietic stem cell compartment, we performed secondary transplants utilizing the cells obtained from primary competitive repopulation assays. We observed an unexpected recovery in engraftment in secondary transplant recipients that was accompanied by an increase in the mRNA expression of Septin8. Since Septin8 and Septin11 are closely related we hypothesized that induction of Septin8 was subserving key functions of Septin11. To test this hypothesis, we created a lentivirus vector that expressed both a shmiR against Septin11 and the Septin8 cDNA. Concurrent overexpression of Septin8 with Septin11 knockdown HSPC caused a significant increase in the total number of colonies compared to controls (Figure 1). These data indicate an important role of Septin11 in murine hematopoiesis and a potential redundant role for Septin8.

Impaired Efficacy and Durability of Hematopoietic Stem Cell Engraftment Caused By Samd9l-Deficiency within Bone Marrow Niches

Germline mutations in the SAMD9 and SAMD9L (SAMD9/L) genes on chromosome 7q drive both childhood myelodysplastic syndrome (MDS)/secondary acute myeloid leukemia (sAML) associated with monosomy 7, and adult-onset MDS/sAML associated with distinct somatic genetic alterations. The ortholog for SAMD9/L in mice is Samd9l. Gain of Function (GoF) and Loss of Function (LoF) Samd9l alleles predispose to MDS at distinct ages. LoF models show that Samd9l-deficient mice, like humans, are more likely to develop MDS during adulthood, while GoF models result in early onset bone marrow (BM) dysfunction and MDS/sAML. Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for bone marrow failure (BMF), MDS, and AML arising from germline genetic syndromes. Our previous research demonstrated that deficits in host BM microenvironment (ME) niches contribute to hematopoietic failure and poor HSC engraftment in murine models of many MDS predisposition syndromes associated with BMF. As we have observed clinical cases where poor engraftment impaired outcomes in patients with SAMD9/L mutations, we recently hypothesized, BM niche functions critical to facilitating HSC engraftment may be impaired due to niche-intrinsic deficits caused by SAMD9/L mutations. Herein, we present data testing this hypothesis, demonstrating Samd9l deficiency alters BM ME composition, extracellular matrix (ECM) regulation, and proinflammatory cytokine signaling resulting in impaired donor HSC engraftment after HSCT. We performed primary (1 ry) and secondary (2 ry) HSCT to model impacts of SAMD9/L deficiency on BM niche function during HSCT using WT ( Samd9l+/+), Samd9l+/-, and Samd9l-/- mice as HSCT recipients of healthy WT GFP + donor BM to measure the capacity of Samd9l LoF niches to engraft donor HSCs. To assess changes in niche composition and gene expression during HSCT, flow cytometry, multiplex ELISA, and RNASeq analysis were performed on WT, Samd9+/- and Samd9-/- mice. Compared to WT control niches, Samd9+/- and Samd9-/- niches exhibited impaired efficiency of donor HSC engraftment at 3 weeks post-1 ry HSCT, as measured by competitive 2 ry-HSCT performed with 1 ry recipient BM harvested at this timepoint. Beyond 30 weeks post-2 ry-HSCT in this model, GFP + reconstitution of GR1 + myeloid, RBC's, and platelets was reduced by 25%, 18%, and 35% in 2 ry recipients of Samd9+/-1 ry recipient BM and by 26%, 28%, and 32% in 2 ry recipients of Samd9-/- 1 ry recipient BM. Durability of HSC engraftment by 8 months was even more significantly impaired in Samd9+/- and Samd9-/- niches, with Samd9-/- mice exhibiting low survival by this timepoint. Competitive 2 ry HSCT performed 8 months post-1 ry HSCT showed reduction in trilineage GFP + blood cells out to 24 weeks post 2 ry-HSCT, including reductions in GFP + Gr1 + myeloid cells by 50% and 83% in 2 ry recipients of Samd9l+/-and Samd9l-/-1 ry recipient BM, respectively, compared to control 1 ry recipient BM. Similar reductions in BM long-term HSC and Lin -Sca-1 +cKit + HSC/progenitor cells in 2 ry recipients of Samd9l-/-compared to Samd9l+/+ 1 ryrecipient BM 8 months post 1 ry-HSCT. Following TBI, Samd9l+/-and Samd9l-/- BM niches contained similar percentages of osteoprogenitors, mesenchymal stem cells, and endothelial cells compared to WT controls. Multiplex ELISA on BM plasma collected 48hrs post-TBI revealed that Samd9l+/-and Samd9l-/-deletion in the BM ME led to markedly reduced expression of IFN-γ (43% and 55% reduction in Samd9l+/-and Samd9l-/-), Eotaxin (33%/36%), GM-CSF (47%/47%), and IL-1a (48%/65%). In contrast, expression of niche cell extracts showed markedly increased levels of IL-17 (4 fold) and CXCL11 (1.4 fold) in Samd9l+/-and of IL-17 (5.3 fold) IL-12p70 (1.3 fold), and CXCL11 (1.5 fold) in Samd9l-/-, suggesting that aberrantly active Th17 responses caused by niche Samd9l deficiency may be responsible for impairing HSC function and maintenance post-HSCT. RNASeq analysis showed downregulation of ECM genes in both Samd9l-/-and Samd9l+/-models. Our data demonstrates BM niche function is impaired by Samd9l deficiency in a gene dosage-dependent manner. These deficits are associated with increased expression of a specific inflammatory cytokine pathway, suggesting that aberrant growth factor receptor recycling due to Samd9l deficiency may drive stress hematopoiesis and alter HSC cell fate decisions leading to failure of durable donor HSC maintenance post-HSCT.

Varicella-associated disseminated intravascular coagulation secondary to Henoch-Schönlein purpura with renal and gastrointestinal system involvement in a child: A case report.

Immunocompromised patients who developed varicella-zoster virus (VZV)-associated disseminated intravascular coagulation (DIC) previously included recipients of bone marrow, hematopoietic stem cell, or organ transplantations, patients with primary nephropathy receiving corticosteroid therapy, cancer patients receiving chemotherapy, and patients with human immune deficiency virus infection. The case reported here is novel because, to our knowledge, there has been no report of VZV-associated DIC after the onset of Henoch-Schönlein purpura (HSP). To report the successful treatment of a novel pediatric case with VZV-associated DIC secondary to HSP. An 8-year-old girl developed VZV-associated DIC 24 days after diagnosis of HSP with renal and gastrointestinal involvement. She was treated with methylprednisolone at a local hospital for 19 days, and suddenly developed fever starting from day 4 in our hospital. Her fever persisted with vesicular skin rashes on her back, strong abdominal and lower back pain, epistaxis, hematochezia, erosion and bleeding on her lips, in her mouth and at puncture sites on day 5. She was diagnosed with DIC with the laboratory evidence of dramatically decreased platelet count and fibrinogen, prolonged activated partial thromboplastin time and prothrombin time, and increased fibrin degradation products including d-dimers. She also developed multiple organ dysfunction syndrome. On day 7, the patient VZV nucleic acid result turned out to be positive. Methylprednisolone treatment was discontinued, and she was given a multi-modality therapy including medications of acyclovir and antibiotics, intravenous gamma-immunoglobulin, various blood product transfusions, continuous renal replacement therapy, plasma exchange, and administration of liver and gastrointestinal system protection drugs. The patient multi-organ function damage gradually recovered. After VZV control, the patient was treated with oral methylprednisolone again for HSP with nephritis. Urine analysis was normal 1 year later, and oral hormone was discontinued. No complication or relapse occurred during 2 years of follow-up. This case report, for the first time, adds HSP treated with corticosteroids to the spectrum of clinical conditions that progressed to life-threatening secondary varicella-associated DIC. Early identification of varicella infection and DIC, combined with timely antiviral, immunoglobulin transfusion, plasma exchange, and other combined therapies are essential for saving patients' lives.

Abstract 14517: apoE Receptor-2-R952Q Variant in Macrophages Potentiates Hyperlipidemia and Atherosclerosis in LDL Receptor-Knockout Mice via Increased Secretion of Soluble LDL Receptor-Related Protein-1

Introduction and objective: Apolipoprotein E Receptor-2 (apoER2) is a type I transmembrane receptor in the LDLR family expressed in vascular smooth muscle cells (VSMC), endothelium, and macrophages. An apoER2 mutation referred to as apoER2-R952Q (R952Q) is associated with elevated plasma triglyceride levels and increased risk of premature coronary artery disease and myocardial infarction in humans. This study investigated the role of the R952Q mutation of apoER2 in modulating disease development. Methods and Results: We generated R952Q-expressing mice using a CRISPR-directed approach. This mouse line was crossed with the LDLR knockout mouse line and the progenies were fed a high cholesterol diet for 16 or 24 weeks. Results showed that unlike apoER2 knockout, the accelerated atherosclerosis R952Q variant mice exhibited was linked to increased plasma lipid levels. Considering apoER2's expression, which is notably absent in hepatocytes, we investigated the impact of R952Q myeloid cells on disease progression via bone marrow transplant. Results showed that wildtype apoER2 mouse recipients of R952Q bone marrow recapitulated the elevated plasma lipid level phenotype while R952Q mouse recipients of wildtype apoER2 bone marrow did not. Due to the absence of LDLR in our model, we investigated LRP1’s role in the context of myeloid-specific R952Q and found an increase in the production of hypercholesterolemia-associated soluble LRP1 (sLRP1) in the plasma of high cholesterol diet-fed mice with myeloid-specific R952Q. sLRP1 may compete with cell-surface LRP1 in lipoprotein clearance, suggesting a novel mechanism by which the R952Q mutation in macrophages may increase plasma lipid levels, thereby accelerating atherosclerosis. Conclusion: The R952Q mutation in apoER2 accelerates atherosclerosis via overproduction of soluble LRP1, a gain-of-function mechanism in macrophages resulting in hypercholesterolemia.

Role of Lama4 Expression in Bone Marrow Niche for the Progression and Treatment Response of Chronic Myeloid Leukemia

Although tyrosine kinase inhibitors (TKIs) have dramatically improved the treatment outcomes of chronic myeloid leukemia (CML), TKIs cannot eliminate CML-initiating stem cells (LSCs), leading to CML persistence and relapse post TKI-discontinuation. Thus, it is imperative to develop more effective therapeutic options to overcome the treatment challenges. Targeting the bone marrow (BM) niche has become one of the research focuses in drug discovery since increasing evidence suggests that BM niches protect CML LSCs. Laminin alpha4 chain (Lama4), a functional chain for several laminin isoforms, is widely expressed in bone marrow (BM). We have recently shown the critical impact of LAMA4 expression in the microenvironment for hematopoiesis regeneration including megakaryocyte maturation and acute myeloid leukemia progression in mice (Cai et al., Blood, 2022). Furthermore, we have by RNA sequencing shown downregulation of LAMA4 mRNA in freshly isolated BM mesenchymal stem cells (MSCs) from newly diagnosed patients with CML (Dolinska, Cai, Mansson, et al., Blood, 2023). However, the functional impact of the LAMA4 expression on CML LSCs remains to be investigated. We have here explored the role of LAMA4 in CML development and drug response by using both mouse models and primary patient BM samples. To first assess the in vivo impact of LAMA4 expression in BM niches on CML development, we transplanted BM cells (CD45.2) from Scl- tTA× TRE-BCR-ABL1 (BCR-ABL1) mice prior or following tetracycline withdrawal into irradiated Lama4+/+ and Lama4-/- CD45.1mice and analyzed CML progression and BCR-ABL1 LSC activity by flow cytometry and droplet digital PCR (ddPCR). Following transplantation of unfractionated BM cells from the BCR-ABL1 mice with CML induced by tetracycline withdrawal, Lama4-/- recipients showed significantly increased platelet counts in blood compared to Lama4+/+ recipients at 2-8 weeks post-transplantation (P=0.005 at week 2, P= 0.008, n=7-8, at week 8), which is in contrast to the impaired platelet recovery in non-transplanted Lama4-/- mice following sublethal irradiation. This suggests that Lama4 deficient microenvironment is favorable for platelet production from BCR-ABL1 CML cells. Consistent with this, megakaryocyte-erythrocyte progenitors (MEPs) derived from BCR-ABL1+ cells were significantly increased in Lama4 -/- BM at the endpoint (8 weeks, p=0.02, n=3). Megakaryocyte progenitors (MkPs) seemed to be increased in Lama4-/- recipient BM (P=0.05, n=3). Intriguingly, when LIN -SCA1 +KIT + CD150 + hematopoietic stem cells from BCR-ABL1 mice without prior tetracycline withdrawal were transplanted, the donor-derived MEPs were significantly reduced in Lama4-/- BM (P=0.02, n=4-6), which is accompanied with a trend of reduction in MkP but no changes in platelets. ddPCR analysis indicated that the ratio of BCR-ABL1 + cell fraction to total CD45.2 + cells (detected by FACS) seemed to be lower in Lama4-/- recipients (33.6%) than that in Lama4+/+ recipients (41.5%), raising a possibility that Lama4-/- niches is less suitable for maintenance of BCR-ABL1 + cells. Together, these data point to a possible differential regulation of CML stem and progenitor cells by the Lama4 expression. More work will be done to dissect the regulatory mechanisms through in vitro co-cultures, transplantation and confocal imaging. In parallel, the functional impact of LAMA4 on human CML LSC survival was evaluated by a revised long-term culture-initiating cell assay using CML patient derived MSCs. The preliminary result indicated that recombinant LAMA4 peptides appeared to inhibit CML patient BM-derived CD34 +CD38 - cell growth. In conclusion, LAMA4 is downregulated in BM MSCs and endothelial cells of CML patients at diagnosis. Lama4-/- microenvironment might act differentially in regulating BCR-ABL1 + hematopoietic stem and progenitor cell activity post-transplantation. However, more work is required to validate the finding and the effect of LAMA4 peptides on the megakaryocyte differentiation and maturation.

IL1RAP-Specific T Cell Engager (TCE) Antibody Efficiently Depletes Acute Myeloid Leukemia (AML) Leukemic Stem Cells (LSCs)

LSCs are self-renewal primitive cells that initiate and maintain AML. Because they are highly therapy resistant, they are often responsible for treatment failures. Due to the lack of a specific antigen (Ag) and their quiescent nature, LSCs have been challenging to target and eliminate. IL1RAP is highly expressed on most human AML cell lines and primary blasts, including LSC-enriched CD34+CD38- cells, but not on normal hematopoietic stem cells (HSCs). To leverage this observation, we generated over 50 unique mouse IL1RAP monoclonal (m) antibodies (Abs), grouped them based on sequence, and selected representative members from the clades to generate murine-human chimeric mAbs. One of the mAbs, termed #24, had the strongest antibody-dependent cell-mediated cytotoxicity (ADCC) properties and the greatest in vivo anti-AML activity. Thus, it was advanced to generate a bispecific (Bs) anti-IL1RAP/CD3 TCE Ab termed BiF002, which incorporates a human IgG1 Fc mutated to lack FcR binding. Incubation of BIF002 with healthy donor T cells and AML cell lines or primary AML blasts induced IL1RAP dependent T cell activation, and Ab-dependent T cell lysis of leukemic cells in a dose-, time- and effector to target (E: T) ratio-dependent manner. The IC 50 against MOLM13 and THP-1 was subnanomolar (0.047nM, 95%CI:0.036-0.060nM and 0.025nM, 95%CI:0.14-0.40nM, respectively), at E:T ratio 5:1 at 48 hours using resting T cells. The IC 50 of IL1RAP pos AML bulk and CD34+ blasts (n=6) was also subnanomolar (0.45nM; 95%CI: 0.28-1.96nM). Co-cultures of normal CD34+ cells with BIF002 and T cells did not activate T cells or eliminate the normal CD34+ cells. To test the antileukemic activity of BIF002 in vivo, we engrafted luciferase (luc)-expressing MOLM13 cells into NSG-SGM3 (NSGS) mice. On day (D) 3, in vitro expanded healthy donors' T cells (3x10 6/mouse, weekly), with either BIF002 or a control IgG (10µg,) were administered intravenously (IV) every 3 days x 4-weeks. Mice treated with T cells + BIF002 exhibited a 100-fold greater tumor reduction on D33 than other groups, as measured by bioluminescence imaging (BLI), and had a significantly longer overall survival (OS; median 54.5 D) compared to IgG (27 D, p=0.0045), BIF002 alone (36 D, p=0.0049), or T cells + IgG (33 D, p=0.0062) treated controls. In initial dose finding studies using a similar schema, lower BIF002 doses (0.1 and 1 µg) were tested. Mice treated with T cells and 1 µg of BIF002 had a 100-fold tumor reduction on D29 and longer OS (median 57 D) compared to T cells + 0.1µg of BIF002 (31 D, p=0.0018) or T cells + vehicle (34 D, p=0.0019). We also engrafted luc-expressing primary blasts from a relapsed/refractory complex karyotype AML patient into NSGS mice that treated for 3 weeks using the above described schema (10µg). In this experiment, we used as control BIF026, a bispecific Ab (BsAb) carrying point mutations in both Fab arms to abrogate target binding to IL1RAP and CD3. Mice treated with T cells + BIF002 exhibited a &amp;gt;1000-fold reduction in disease burden and longer OS than BIF026 or BIF002 alone or T-cells+ BIF026-treated mice (median 46 D vs 26, 25 or 30 D, respectively, all comparisons, p=0.0003). In a second patient-derived xenograft (PDX) model, blasts from a relapsed complex karyotype AML patient were engrafted into NSG mice. Treatment was initiated on D10, with T cells given weekly along with 10µg of BIF002 or BIF026 for 3 weeks. After 70 days, all mice treated with T cells + BIF002 were alive, with no detectable blasts in peripheral blood or bone marrow (BM), while all vehicle (median 38 D, p=0.0002) and T cells + BIF026 (38 D, p=0.0001) treated controls had died of leukemia. To determine if T cells + BIF002 eradicated LSCs, we randomly selected 3 female mice from each group on D 34 and transplanted BM mononuclear cells (MNC) into secondary NSGS recipients (n=7). At D 90, no recipients of BM from donors treated with T cells + BIF002 had detectable blasts and remained alive, while all vehicle (median 26 D, p=0.0004) and T cells + BIF026 (median 26 D, p=0.0002) treated controls succumbed to AML. Importantly, no obvious hematopoietic or non-hematologic side effects were observed after BIF002 administration alone or with activated T cells in normal and leukemic mice. In summary, we provide evidence of the subnanomolar potency and efficacy of BIF002, a novel anti-IL1RAP/CD3 TCE, in eliminating AML blasts, including LSCs. BIF002 is currently undergoing IND-enabling studies.

In Vivo Antileukemic Activity of Ost-01 in Acute Myeloid Leukemia (AML): A Novel Natural Product (NP) from Baccharis Coridifolia

Baccharis is the largest genus in the Asteraceae and has been long used by phytomedicine practitioners in South America to treat a variety of medical conditions (i.e., wounds, infections, cancers). Herein, we report on the antileukemic activity of OST-01, a novel NP from Baccharis coridifolia. OST-01 was obtained through maceration of Baccharis coridifolia leaves in ethanol; the starting doses for testing the OST-01 activity against leukemic cells were derived empirically from routinely used dosages of similar plant extracts. OST-01 (0.25-2 µL/mL) inhibits cell growth and induces apoptosis of AML cell lines (MV-4-11, KG-1a, Kasumi-1, and HL-60) and primary CD34 + CD38 ‒ AML blasts [enriched for leukemia stem cells (LSCs)] but did not affect normal human CD34 + CD38 ‒ mononuclear cells [MNCs, enriched for hematopoietic stem cells (HSC)]. Compared to vehicle (V; 1 µl/g/BID PO x 21 days), OST-01 (1 µl/g/BID PO x 21 days) reduced leukemia burden (WBC: 59.4 vs 96.4x10 3/µL, p=0.01; spleen size: 0.38 vs 0.77 g, p&amp;lt;0.0001) and prolonged survival (median: 123 vs 92 days, p&amp;lt;0.0001) of MllPTD/WT/ Flt3ITD/ITD AML mice. In secondary transplant experiments, recipients of bone marrow (BM) MNCs from OST-01-treated donors, lived significantly longer than the recipients of BM MNCs from V-treated donors (median: 58 vs 26 days, p&amp;lt;0.0001), suggesting that OST-01 reduced LSC burden. We confirmed these results in a Cbfb-MYH11 ( CM) knock-in AML mouse model. OST-01-treated mice had a longer survival than V-treated controls (median: 70 vs 58 days, p&amp;lt;0.0001) and secondary recipients of BM MNCs from OST-01-treated donors lived also significantly longer than those of BM MNCs from V-treated donors (median: 63 vs 38 days, p&amp;lt;0.0001). Similar results were also observed in mice engrafted with luciferase-expressing FLT3-ITD+ MOLM-14 AML cells. Bioluminescence imaging indicated that OST-01 reduced leukemic burden compared to V treatment (@day 30: 2.3 vs 4.5x10 6 photons/s/cm 2/sr, p&amp;lt;0.0001); OST-01-treated mice lived significantly longer than V-treated controls (median: 46 vs 32 days, p&amp;lt;0.0001). Of note, no OST-01-related toxicity given within a dose range of 1-3 µl/g/BID, PO x 7, 28 or 56 days was observed in normal B6 mice (n=120 mice). Compared with OST-01 alone or V, combination of OST-01 and the BCL-2 inhibitor, venetoclax (VEN) demonstrated a synergistic activity on AML cell lines and primary blasts (max synergy score: 28.16), and prolonged survival of MllPTD/WT/ Flt3ITD/ITD AML mice (VEN/OST vs OST and VEN, median: 156 vs 123 and 95 days, p=0.0003 and p&amp;lt;0.0001; secondary transplant median: 105 vs 58 and 31 days, p&amp;lt;0.0001 and p&amp;lt;0.0001, respectively) and CM AML mice (VEN/OST vs OST and VEN, median: 85 vs 70 and 58 days, p=0.0005 and p&amp;lt;0.0001; secondary transplant median: 95 vs 62.5 and 48 days, p=0.0003 and p&amp;lt;0.0001, respectively). To gain insights into the mechanism of action of OST-01, we performed RNA-seq on OST-01- or V-treated HL-60 cells and CD34+CD38- AML blasts. By GSEA analysis, we identified 50 enriched Hallmark gene sets (26 upregulated and 24 downregulated) in OST-01- vs V-treated HL-60 cells and 49 (32 upregulated and 17 downregulated) in OST-01- vs V-treated CD34+CD38- AML blasts. The Hallmark gene sets comprising Myc targets were among the most downregulated in OST-01-treated cells. We confirmed these results showing an OST-01-dependent decrease in c-myc, inhibition of c-myc signaling, and deregulation of c-myc-dependent nucleolar structure and ribosome biogenesis in HL-60 cells and CD34+CD38- AML blasts. To this end, OST-01 enhanced ubiquitination and degradation of c-myc and nucleolar proteins (NPM1, NS, nucleolin) and in turn induced nucleolar disruption as shown by immunoblotting, immunoprecipitation, and electron microscope imaging in HL-60 cells and CD34+CD38- AML blasts. Of note, OST-01 also inhibited ErbB3 binding protein 1 (Ebp1)-enhanced ribosomal RNA (rRNA) synthesis through upregulation of expression and function of RNA Polymerase I (Pol I) and DEAD-box helicase 21 (DDX21). In summary, we provide the first report of a significant in vivo antileukemic activity of a novel NP, OST-01. The mechanisms of action of OST-01 are mediated by inhibition of c-myc-dependent signaling, rRNA synthesis and ribosome biogenesis. To date, no preclinical toxicity has been observed. We concluded that OST-01 is a non-toxic, potentially new therapy for AML; translation from the bench to the bedside is underway.

Decline in Cardiolipin in Hematopoietic Stem Cell during Aging Alters Their Regenerative Potential

Hematopoietic Stem Cells (HSCs) are known for their regenerative potential. This property allowed the use of HSC in bone marrow transplantation to treat hematological disorders such as leukemia. However, aging influences these characteristics of HSCs. The major hallmarks of aging are limited self-renewal and reconstitution capacity resulting in HSC exhaustion in aged individuals. Mitochondrial metabolism and activity are active drivers of HSC fate decisions and data from our lab shows that mitochondria in aged HSCs have increased sphericity with a polarized mitochondrial network and a lower mitochondrial membrane potential. An important aspect of mitochondrial functions is the lipid composition of their membranes. A lipid trafficking assay showed an atypical pattern of lipid incorporation by mitochondria in aged HSCs suggesting that mitochondrial lipids become abnormal upon aging. The overall content of Cardiolipin (CL) which is a signature mitochondrial lipid, found exclusively in the inner mitochondrial membrane was also reduced during aging. This was done with flow cytometry using a CL marker NAO (Non-Acridine Orange). Tafazzin encoded by the gene TAZ is crucial for remodeling CL into its mature form which is required for maintaining mitochondrial functions. Mutations in the TAZ gene have been known to cause a reduction in the synthesis of mature CL which is often associated with HSCs functional defect. Western Blot analysis showed a considerable decrease in Taz protein expression in aged stem and progenitor cells (HSPC) compared to young HSPC. To understand the effect of reduced Taz expression on HSC functions, we used a doxycycline inducible, sh-RNA mediated TAZ knock-down (KD) mouse model. Bone marrow analysis indicates that the frequency of HSC and multipotent progenitors decreases significantly in 6-month-old TAZ KD mice compared to wild type (WT) mice. Our data shows that following 5FU induction TAZ KD mice have a significant reduction in the multipotent progenitor and HSC population on day 10 and day 21 post 5FU, indicating TAZ KD decrease the regenerative potential of HSCs under stress conditions. Further pointing towards the significance of cardiolipin in HSC regeneration. TAZ KD mice also have abnormal peripheral blood recovery post 5FU mainly affecting the platelets. We next examined the repopulation potential of HSCs isolated from TAZ KD mice using serial competitive transplantation assays. While primary transplanted mice of TAZ KD HSCs exhibited similar donor-cell chimerism compared to mice transplanted with WT HSCs, about 50-60%, secondary recipient of TAZ KD cells had significantly lower donor-cell chimerism than secondary recipients of WT cells. Further, donor-derived HSC frequency in the bone marrow of recipients of TAZ KD HSCs 24 weeks following engraftment was also significantly lower than recipients of WT HSCs. Hence, Taz-deficiency causes a defect in HSC self-renewal under regenerative conditions. We next evaluated whether restoring cardiolipin levels in mid-aged HSCs could rescue their functions. For this, mid-aged HSC were incubated for 3 days under conditions that maintain HSC functions in vitro with Alcar (Acetyl-L-Carnitine), which promotes CL production. In lipid incorporation assay, Alcar supplementation restored mitochondrial membrane potential, mitochondrial organization and lipid incorporation of mid-aged HSCs. In competitive transplant assay, while mid-aged WT HSC treated with vehicle gave rise to a myeloid-bias graft, Alcar supplemented mid-aged WT HSC gave rise to a more balanced graft with increased ratio lymphoid to myeloid cells in the peripheral blood 16 weeks following transplantation, albeit the rescue was partial. This study indicates that HSC mitochondrial membrane lipids become abnormal with aging which causes a decline in HSC function and highlights the critical role of mitochondrial cardiolipin in HSC functions. It implies that metabolite supplementation is a viable option for restoring age-related HSC functional defects.

FRI379 The Gonadotoxic Effects Of Cyclophosphamide And Busulfan On Ovaries In CD-1 Mouse Model

Abstract Disclosure: M. Thonen: None. H. Jiang: None. Y. Luan: None. R. Dong: None. A. Wadhwa: None. S. Kim: None. Hematopoietic stem cell transplantation (HSCT) involves the transfer of healthy donor stem cells to replace a recipient's dysfunctional bone marrow. It can be used for both malignant diseases (eg lymphoma or leukemia) and non-malignant diseases (eg sickle cell anemia). Chemotherapy and/or radiation are usually required to deplete the recipient's bone marrow before transplant, also known as conditioning regimens. Alkylating agents including cyclophosphamide (CY) and busulfan (BU) are frequently involved in these regimens. Primary ovarian insufficiency in young female patients secondary to conditioning regimens presents a challenging fertility complication. Studies show that there may be unbalanced ovarian hormonal levels and decreased ovarian function in young female cancer survivors, however, limited studies have explained the mechanism of medication-induced ovarian toxicity in an animal model. This study aimed to investigate the gonadotoxic effects of CY and BU in CD-1 strain mice and to delineate the underlying mechanism of the gonadotoxic outcomes, eventually suggesting possible fertoprotectants for HSCT recipients’ fertility preservation.Sixteen one-month-old female CD-1 mice (n=4/group) were subcutaneously injected with solvent, BU, CY, or BU+CY, respectively, over the course of six days. The doses (144 mg/Kg BU and 300 mg/Kg CY) and injection plan were adjusted to clinical doses. One week after the last administration, the ovaries and female reproductive tracts were harvested. The samples were embedded in paraffin, then continuously sectioned into 5 μm thickness. The specimen was stained with hematoxylin and eosin (H&amp;E) and TUNEL assays. Different stages of follicles were counted individually by two team members. The serum levels of anti-Mullerian hormone (AMH), 17-β-estradiol, and follicle-stimulating hormone (FSH) were measured. Additional immunofluorescence staining for AMH, inhibin α, and Ki-67 was also performed. All mice survived treatment and appeared grossly normal. The average ovary weight was significantly lower in BU-based treatment groups compared to control and CY alone groups. The primordial (p&amp;lt;0.0001) and primary follicles (p=0.01) in BU, CY, and BU+CY groups were lower than the control group. The average AMH level in the BU-based treatment groups was also significantly lower than in the control and CY-alone group. Our results indicate CY and BU regimens individually were gonadotoxic, reducing the ovarian reserve in CD-1 mice with a synergistic toxic effect when used in combination. Decreased AMH reveals the conditioning regimens are toxic to granulosa cells and the ovarian reserve. Future studies are needed to elaborate possible pathways of chemotherapy-induced ovarian follicle death and the toxicities of other regimens to improve fertility outcomes and better care for female patients receiving HSCT. Presentation: Friday, June 16, 2023

Engineered Knockout of TRPA1 Inhibits Laser-Induced Choroidal Neovascularization Along With Associated TGFβ1 Expression and Neutrophil Infiltration

We examined the effects of gene ablation and chemical inhibition of transient receptor potential ankyrin 1 (TRPA1) on the growth of experimental argon laser–induced choroidal neovascularization (CNV) in mice. CNV was induced in the eyes of 6- to 8-week-old TRPA1-null (knockout [KO]) and wild-type (WT) mice by argon laser irradiation. Gene expression analysis was performed in laser-injured tissues at days 1 and 3. CNV growth was evaluated at day 14. Reciprocal bone marrow transplantation was performed between each genotype to identify the components responsible for either recipient tissue or bone marrow–derived inflammatory cells. Our results show that laser irradiation successfully induced CNV growth at the site of laser injury. The size of induced CNV was significantly smaller in KO mice than in WT mice at day 14, as determined by angiography with fluorescein isothiocyanate-dextran. Invasion of neutrophils, but not macrophages, was suppressed in association with suppression of the expression of transforming growth factor β1 and interleukin 6 in laser-irradiated KO tissue. Bone marrow transplantation indicated that the genotype of the recipient mouse, but not of inflammatory cells, is attributable to the KO phenotype. Systemic administration of a TRPA1 antagonist also reduced the CNV in a WT mouse. In conclusion, TRPA1 signaling in local cells is involved in growth of laser-induced CNV. The phenotype was not attributable to vascular endothelial cells and inflammatory cells. Blocking TRPA1 signal may therefore be a potential treatment strategy for CNV-related ocular diseases.

Evaluation of GenDx Protocol for HLA NGS Genotyping Using the Ion Torrent Sequencing Platform

Abstract Background: The Human Major Histocompatibility Complex (HLA) is the most polymorphic region of the human genome and encodes molecules with a central role in antigen-specific immune responses. Class I and class II HLA genes have an important role in bone marrow and solid organ transplantations. HLA typing by Next Generation Sequencing (NGS) provides unambiguous second field or allelic level resolution and, therefore, better matching of donor and recipient and a higher chance of survival for transplanted patients. The aim of this study was to validate GenDx NGS HLA typing protocol for 6 HLA loci (HLA-A, -B, -C, DRB1, -DQB1 and DPB1). Materials and methods: 240 samples of both bone marrow healthy donors from the Bulgarian Bone Marrow Donors Registry (BBMDR) and bone marrow recipients were sequenced on the Ion Torrent System using the GenDx NGS HLA typing kit, compared to 65 samples from the BBMDR analyzed by Holotype HLA kit (Omixon) and sequenced on the Illumina platform MiniSeq. A number of metrics including allele balance, read length, mappability, coverage and ambiguity were assessed in order to evaluate the GenDx NGS HLA typing protocol. We also investigated haplotype and allele frequencies of class I (HLA-A, -B, -C) and class II (HLA-DRB1, -DQB1 and -DPB1) alleles. Results: The results demonstrate the accuracy of this protocol, eliminating almost all ambiguities, providing a reasonable read length of 180, 76% mappability and 1267 depth of coverage on average for all 6 HLA loci. We found that the most frequent alleles for class I are HLA-A*02:01:01 (27,5%), B*51:01:01 (16,5%), C*07:01:01 (16,5%) and class II – DRB1*11:04:01 (14,5%), DQB1*03:01:01 (26,9%) and DPB1*04:01:01 (33,2%) which corresponds with other investigations of the research team on HLA diversity in the Bulgarian population. Conclusion: The main advantages of GenDx NGS HLA typing protocol are the shorter time for DNA library preparation and the shorter sequencing time. Phasing ambiguities are also liminated, which is a significant advantage in the clinical environment. The presented data show the suitability of this NGS protocol for clinical practice, especially in HLA typing of donors from Bone Marrow Donors registries.