Bone Marrow Niche Research Articles

Overcoming BCR::ABL1 dependent and independent survival mechanisms in chronic myeloid leukaemia using a multi-kinase targeting approach

BackgroundDespite improved patient outcome using tyrosine kinase inhibitors (TKIs), chronic myeloid leukaemia (CML) patients require life-long treatment due to leukaemic stem cell (LSC) persistence. LSCs reside in the bone marrow (BM) niche, which they modify to their advantage. The BM provides oncogene-independent signals to aid LSC cell survival and quiescence. The bone-morphogenetic pathway (BMP) is one pathway identified to be highly deregulated in CML, with high levels of BMP ligands detected in the BM, accompanied by CML stem and progenitor cells overexpressing BMP type 1 receptors- activin-like kinases (ALKs), especially in TKI resistant patients. Saracatinib (SC), a SRC/ABL1 dual inhibitor, inhibits the growth of CML cells resistant to the TKI imatinib (IM). Recent studies indicate that SC is also a potent ALK inhibitor and BMP antagonist. Here we investigate the efficacy of SC in overcoming CML BCR::ABL1 dependent and independent signals mediated by the BM niche both in 2D and 3D culture.MethodsCML cells (K562 cell line and CML CD34+ primary cells) were treated with single or combination treatments of: IM, SC and the BMP receptors inhibitor dorsomorphin (DOR), with or without BMP4 stimulation in 2D (suspension) and 3D co-culture on HS5 stroma cell line and mesenchymal stem cells in AggreWell and microfluidic devices. Flow cytometry was performed to investigate apoptosis, cell cycle progression and proliferation, alongside colony assays following treatment. Proteins changes were validated by immunoblotting and transcriptional changes by Fluidigm multiplex qPCR.ResultsBy targeting the BMP pathway, using specific inhibitors against ALKs in combination with SRC and ABL TKIs, we show an increase in apoptosis, altered cell cycle regulation, fewer cell divisions, and reduced numbers of CD34+ cells. Impairment of long-term proliferation and differentiation potential after combinatorial treatment also occurred.ConclusionBMP signalling pathway is important for CML cell survival. Targeting SRC, ABL and ALK kinases is more effective than ABL inhibition alone, the combination efficacy importantly being demonstrated in both 2D and 3D cell cultures highlighting the need for combinatorial therapies in contrast to standard of care single agents. Our study provides justification to target multiple kinases in CML to combat LSC persistence.

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<0.001) compared to pre-transplantation. In contrast, CD9 expression was significantly downregulated on BM CD34+ cells from 73.4% to 33.5% ( P<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<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.

Hematopoietic Stem and Progenitor Cells Acquire an Inflammatory Memory of the Acute Myeloid Leukemia Niche

Recent studies suggest that acute myeloid leukemia (AML) generates inflammation in the bone marrow (BM) niche to an extent that compromises the function of healthy hematopoietic stem and progenitor cells (HSPC) and impacts patient prognosis. AML xenograft studies have identified several pro-inflammatory mediators (IL-6, IL-1b) that are secreted into the BM niche by leukemic blasts. Prior work by our laboratory also showed contributions involving other AML-secreted factors, in regulating HSPCs in the leukemic niche. HSPC are cellular components of the innate immune system, but the full extent of their inflammatory adaptation by AML is unknown. To overcome some of the experimental constraints inherent in the xenograft approach, we first established non-conditioned congenic grafts of C1498 murine AML cells in C57B/6 recipients. Assessment of the BM revealed that, even at low AML burden, these animals contained a range of elevated pro-inflammatory factors. Single cell RNA-Seq studies of healthy HSPCs, obtained concurrently at sacrifice confirmed an inflammatory transcriptional signature in HSC and MPP populations, with GSEA enrichment for interferon-alpha and -gamma signaling pathways. These observations led us to hypothesize that AML-experienced (inflammation-primed) healthy HSPCs may acquire a durable long-term inflammatory memory that shapes their response to subsequent stimulation during relapse or infection. To test this hypothesis, we adoptively transferred FACS-sorted healthy AML-experienced HSPCs from C1498-engrafted mice (HSPC AML) into secondary recipients. HSPCs transplanted from PBS-injected recipients (HSPC PBS) serve as controls. To assess whether HSPC AML would respond to immune challenge more aggressively compared to HSPC PBS, we exposed these animals with a single-injection of lipopolysaccharide (LPS), followed by tissue harvest and analysis after 24 hours. Bulk RNA-Seq analysis of HSPC AML from the LPS-challenged recipients showed amplified GSEA signatures for tumor necrosis factor- and NF-kB signaling pathways compared to HSPC PBS. In addition, LPS-challenged HSPC AML showed amplified expression by several krüppel-like factors (KLF) genes. Analysis for transposase-accessible chromatin loci (ATAC-Seq), concurrently obtained from LPS-challenged HSPC AML echoed the enrichment of KLF motifs as well. Collectively, the C1498 model suggests that initial AML exposure programs HSPC AML for an amplified inflammatory and differentiation program. To further ascertain disease relevance and reproducibility we turned to a doxycycline-inducible (i)MLL-AF9 AML model. Given the low leukemic burden in the BM of C1498-grafted animals, we wanted to test the hypothesis that secreted factors accounted, at least in part, for the inflammatory memory. Indeed, injection of AML secreted factors from iMLL-AF9 models revealed a strong inflammatory signature in bulk RNA-Seq studies. To determine if innate immune priming in HSPC can be directly mediated by AML crosstalk, we exposed ex vivo-expanded HSPCs to purified secreted factors (SF AML) from (i)MLL-AF9 AML blasts, followed by a secondary inflammatory challenge with LPS seven days later. Gene expression analysis of SF AML-experienced LPS-challenged HSPCs confirmed myeloid-skewed progenitor differentiation and increased expression of inflammatory targets. Together, our data show for the first time that phenotypically normal, AML-experienced HSPCs carry a durable inflammatory legacy whereby primary exposure to AML secreted factors alters their response to secondary stimuli. Our experiments phenocopy observations of trained immunity in other experimental systems, and suggest the acquisition of an innate immune memory in the AML niche. Immune priming may impact the role of these HSPC in the context of inflammation associated with aging, or clones with predisposing molecular lesions such as TET2 or DNMT3A.

Trafficking Kinetics of Steady State Versus Mobilized Hematopoietic Stem and Progenitor Cells in Non-Human Primates

The safety and efficacy of hematopoietic stem and progenitor cell (HSPC) allogeneic transplantation and autologous gene therapies are dependent on mobilization of HSPCs out of bone marrow (BM) niches into peripheral blood (PB) and homing of infused HSPCs into open niches. However, the disposition of HSPCs exiting the marrow in steady state and following mobilization are not well understood. We characterized short-term (≤ 48 hours (h)) HSPC dynamics during steady state and following mobilization (single dose AMD3100 (plerixafor), 1mg/kg) in rhesus macaques (RMs), a model with direct preclinical relevance. We used serial intravascular staining (SIVS) (Potter et al., 2021; Morlock et al., 2021), based on timed intravenous infusions of multiple fluorescently labeled anti-CD45 antibodies (Ab) (half-life ~8.5 min), to specifically and instantaneously label all blood leukocytes at the time of each infusion, providing a window into blood residence time and tissue entry and egress. SIVS Abs are stably retained on the surface of leukocytes for up to one week and persist with migration into tissues, allowing determination of the passage of cells in and out of circulation. SIVS Abs were administered at multiple time points over 48 h, and PB was collected 5 (minutes (min)) before and 5 min, 2 h, and 4 h after each infusion. PB and BM were collected 5 min after the final Ab infusion, which was used to designate cells as intravascular (-5 min +/IVas +) or tissue-homed (-5 min -/IVas -). HSPC subsets and SIVS Abs were analyzed via flow cytometry. Additionally, trafficking to extramedullary HSPC tissue reservoirs (spleen, thymus, lymph nodes) was studied in a RM euthanized immediately following the final Ab infusion. Under steady state (n=3 RMs), we documented rapid (≤ 2 h) homing of ~55-95% of blood HSPCs out of the circulation to alternate tissue reservoirs. Of the circulating HSPC subsets, no significant differences were observed between the trafficking of HSC-enriched (CD34 +CD45RA -CD90 +), lympho-myeloid progenitor-enriched (CD34 +CD45RA +CD90-), and multipotent and erythro-myeloid progenitor-enriched (CD34 +CD45RA -CD90 -) HSPCs (Radtke et al., 2020). Following 48 h, minimal (≤ 10%) circulating HSPCs from all earlier times were found in BM, suggesting that steady state circulating HSPCs rarely “rehome,” at least within 48 h (Figure 1). Of the extramedullary reservoirs studied, minimal HSPCs trafficked to the thymus or lymph nodes; however, the spleen had a population (< 14 %) of HSPCs moving in and out over a 24 h period. To investigate the impact of AMD3100 on HSPC trafficking (n=1 RM), four SIVS Abs were administered 5 min prior to mobilization (baseline), at peak of mobilization (4 h post-AMD3100), and 10 h and 12 h post-AMD3100, as the concentration of blood HSPCs decreased. PB was collected 5 min before and 5 min, 0.5 h, and 2 h post each SIVS Ab infusion to monitor HSPC kinetics; PB and BM were collected at 12 h post-AMD3100, and final PB was collected at 24 h post-AMD3100. Compared to steady state HSPC dynamics described above, mobilized HSPCs labeled at the peak of mobilization exhibited prolonged circulation (Figure 2), with a higher portion of the mobilized cells remaining in the blood at the 2 h time window compared to steady state. However, there was no increase in the fraction of the HSPCs rehoming to the BM (Figure 1), suggesting that mobilized HSPCs do not exhibit a greater propensity for rehoming compared to steady state and that BM niches are still open 12 h after mobilization. The extended availability of these niches holds potential relevance for the use of potent mobilizing regimens to facilitate engraftment of transplanted autologous gene-modified or allogeneic HSPCs. In summary, under both steady-state and mobilization conditions, HSPCs exhibit minimal rehoming to bone marrow niches, and mobilized HSPCs linger in circulation for up to 24 h post-mobilization. Further studies in progress aim to investigate longer term BM rehoming and better define extramedullary sites of HSPC egress at steady state or following mobilization. Harnessing knowledge of HSPC dynamics holds potential to improve mobilization approaches and provide key insights into developing efficient mobilization-based conditioning for safer HSPC transplantation.

H2-K1 on MLL-AF9 Leukemia Cells Facilitates the Escape of NK Cell-Mediated Immune Surveillance

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults, and prognosis is poor; 5-year survival approximately 30%. Recently, there is an emerging recognition of the innate immune system, in particular Natural killer (NK) cells and macrophages, for immune surveillance against AML, but the mechanistic basis for this is mostly unknow. Identifying how AML cells escape immune surveillance by NK cells may translate into new treatment opportunities for AML patients. To identify new therapeutic opportunities, we recently performed an in vivo CRISPR/Cas9 screen targeting 961 cell surface genes using the MLL-AF9 AML mouse model. One of the top leukemia stem cell dependencies in the bone marrow niche was H2-K1, an ortholog of human HLA-A, a classical MHC class-I molecule. In validation experiments, we observed a seven-fold depletion (p<0.001) of H2-K1 sgRNA-expressing c-Kit +Cas9 + leukemia cells in the bone marrow. In contrast, genetic disruption of H2-K1 did not impact the growth and survival of MLL-AF9 leukemia cells in culture. Given the known suppressive role of MHC class-I molecules for immune cells, we speculated that H2-K1 mayprovide inhibitory signals that counteract immune-surveillance mechanisms in the bone marrow niche. To test this hypothesis, we depleted NK cells and macrophages prior to transplantation of the c-Kit + MLL-AF9 leukemia cells. Macrophage depletion by clodronate liposomes did not affect the in vivo expansion of H2-K1 sgRNA-expressing leukemia cells demonstrating that macrophages were not suppressed by H2-K1 on the leukemia cells. In contrast, NK1.1 antibody-mediated depletion of NK cells fully rescued the depletion of H2-K1 sgRNA-expressing leukemia cells in vivo. These findings suggest that H2-K1 expression on MLL-AF9 leukemia cells inhibits NK cells in this model. Consistent with these findings, H2-K1 knockdown in leukemia cells triggered a two-fold increase of INFγ production (p<0.01) in the NK cells, accompanied by augmented apoptosis of the leukemia cells (p<0.01). Given that NK cells have been shown to be dysfunctional in AML patients, we next explored whether leukemia development affects NK cell maturation. The expansion of leukemia cells in the mice skewed NK cells towards a M1 (CD27 +CD11b -) state and decreased the level of the more cytotoxic M2 (CD27 +CD11b +) and M3 (CD27 -CD11b +) populations. H2-K1 disruption in the leukemic cells restored the level of M2 and M3 NK cell population in the bone marrow. Notably, restoration of matured NK cell populations was accompanied by an increased expression of NKG2D, an activating receptor, indicating a more cytotoxic state of the NK cells. In line with these findings, ablation of H2-K1 in leukemia cells induced JAK/STAT and NF-κβ signaling in the NK cells. Taken together, our study identifies that H2-K1 on MLL-AF9 leukemia stem cells facilitates immune evasion by suppressing NK cells. H2-K1 alters the maturation and activation of NK cells in the bone marrow niche. These findings increase our understanding of how leukemia cells escape immune surveillance and suggest that the identification of corresponding mechanisms in human AML could pave the way for new therapies that boost the endogenous NK cells by restoring immune surveillance mechanisms.

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.

The Bone Marrow-Targeted Conditioning Stress Test Demonstrates Vertebrae Niche Being More Radio-Tolerant Than Femur, Indicating Skeleton Resilience and Global Heterogeneity

Introduction: Myeloablative conditioning prior to Hematopoietic cell transplant, the curative treatment for several hematopoietic disorders, is essential for successful donor cell homing and engraftment. Despite the anatomical classifications, it has been challenging to grasp the complexity of large skeletal systems' global and local bone marrow (BM) environments, which may play a crucial role in donor homing, engraftment, and treatment response. Moreover, due to a lack of comprehensive in vivo studies, there is sparse information about radio-sensitivity on BM niches in local (i.e, endosteal and sinusoidal) and regional skeletal systems (femur, spine etc.,). This may be due to a lack of technology to deliver targeted radiation and therefore using systemic whole-body radiation which resulted in lethality at higher doses due to the composite effect of hematopoietic failure and organ toxicity. Hence, we developed a preclinical image-guided total marrow irradiation (TMI) model, which allows BM specific increase in radiation dose while sculping dose to vital organs, therefore providing an opportunity to evaluate the radio-sensitivity of the BM niche. Here, using TMI dose escalation, we determined the radiation sensitivity of BM niches and its effect on donor homing. Methods: TMI treatment delivery was developed as described previously (Zuro et al., 2022). Different radiation doses (12, 16, 20 and 24 Gy) were delivered to the host (CD45.1 B6 mouse) skeleton in two fractions and was transplanted with 10 million Donor BM cells (CD45.2 B6). The femur and L Spine were harvested for these treated mice at 20+ h, post BMT. The central marrow (CM) cells were harvested by flushing/gently crushing the bone with Collagenase I and Dispase I enzymatic solution, while endosteal marrow (EM) was harvested by digesting the bone fragments with Collagenase IV enzyme, for 30 minutes at 150-200 rpm at 37° C. The isolated cells from endosteal and Central marrow were stained with standard verified markers for Hematopoietic stem cells, Mesenchymal Stem cells and Endothelial cells (18 antibody Panel) and data was acquired in Aurora spectral flow cytometer (Cytek) and was analyzed using FloJo software. Results: The CM cells were more sensitive to radiation, ~≤10% cell survived compared to untreated controls (20h post BMT) while EM cells were more radio tolerant both in Femur (~≥30% survived) and L spine (≥80% survived), however the endosteal cells from l-spine were more radio tolerant than Femur (~≥ 80% vs 16-28%) ( Fig 1 A-B). Further, in femur it was dose dependent i.e., cell number reduced with increasing doses (~28% (12 Gy) to ~16% (24 Gy), unlike l-spine where cell numbers were similar across treated doses. Also, in femur, the Donor cells preferred EM niche over CM, however at higher dose (24 Gy) this spatial preference was lost ( Fig 1C), unlike l-spine which was similar in CM and EM at all doses. The Sca1 + MSC was significantly higher in EM than CM both in the femur and l-spine, however it decreased in femur at higher doses (24 Gy), while remained unchanged in l-Spine ( Fig 1D-E). The arteriolar endothelial cells (AECs) were predominantly present in the EM both in femur and spine, and while sinusoidal endothelial cells (SEC) were higher in EM than CM in Femur, however Spine did not show this pattern. Although, at higher doses (24 Gy), the AECs were significantly reduced even in the EM but were still higher than CM ( Fig 1F). The donor HSPCs (Lineage- cKit+ cells) were significantly higher in the central marrow while the HSCs (Lineage-, cKit+ Sca1+, CD150+) were higher in endosteal marrow. Only in femur, at higher doses AECs, SECs, and Sca1+ MSCs were reduced, resulting in lower CD150+ HSC homing, which was not affected in the l-spine ( Fig 1G-H). Conclusion: In summary, skeletal niche tolerates much higher radiation than previously performed using conventional myeloablative studies and support donor homing. Increasing radiation doses reduced the BM niche constituent cells like AECs, SECs and Sca1+ MSCs, preferentially in femur and reducing homing, surprisingly though the spine BM was more resistant and did not show any reduction in above mentioned cellular composition. This preferential radiotolerance between skeletal regions warrants more studies to understand the' BM stromal cells radio sensitivity with respect to regional and spatial skeletal niche.

The Aging Bone Marrow Niche Disrupts Glutamine Metabolism By Upregulating HSC Derived Glutamine Synthetase

As our aging population grows it is becoming increasingly important to better understand the physiological changes that drive that drive the aging process. Aging has long been known to drive haematopoietic stem cell (HSCs) dysfunction and therefore has been implicated in promoting the development of age-related haematopoietic diseases, such as leukaemia and myeloma. It is likely that a combination of intrinsic changes within the HSCs and extrinsic changes within the bone marrow (BM) microenvironment contribute to HSC aging. We have previously shown that the bone marrow niche accumulates senescent cells which contribute to impaired HSC response to stress 1. Here we investigate the intrinsic changes that can be observed in aged HSCs and how these are influenced by the bone marrow niche. In order to investigate the intrinsic changes that drive metabolic HSC aging we BM was isolated from young (8-12 weeks) and aged (18-24 months). Single HSCs were then FACS purified and sequenced using the SmartSeq2 protocol. Three transcriptionally distinct populations were identified, HSCs from young mice (Y), HSCs from aged mice with an aging transcriptional signature (O) and HSCs from aged mice that are transcriptionally similar to HSCs from young mice (OY). Assessment of differentially expressed genes between these three populations revealed that glutamine synthetase ( glul) is significantly upregulated in the O population compared to both the Y and OY population. Glul is responsible for the conversion of glutamate to glutamine and therefore levels of both were next measured in FACS purified HSCs from aged and young mice. Results show that whilst there was no difference in glutamate levels, the glutamine levels were significantly reduced in HSCs from aged mice. These data suggest that glutamine metabolism is impaired in aged HSC. Next, to determine the impact of the aged BM niche on glul expression in aged HSCs, HSCs from young and aged PepCboy (CD45.1+) mice were FACS purified and adoptively transferred into young C57Bl/6 mice. After 12 weeks the mice were sacrificed and engraftment of CD45.1+ cells was confirmed by flow cytometry. In addition CD45.1+ HSCs were FACS purified to assess glul expression and measure glutamine and glutamate levels. Results confirm that the mechanism for changes in glul expression and glutamine levels is driven by the aging BM niche and transplantation into a young niche reverses this change. To understand the importance of dysregulated glutamine metabolism in aged humans we collected BM from older patients undergoing elective hip replacement surgeries. All samples were collected following informed consent and under approval of the UK NHS Health Research Authority (ref07/H0310/146). CD34+ cells were isolated and RNA extracted to measure glul expression. Similarly, to data from aged mice glul expression was elevated in aged CD34+ cells compared to young CD34+ controls. Furthermore, glutamine levels were also low in aged CD34+ cells. This suggests that the changes in HSCs from aged mice correlate with finding in human stem cells and mice are therefore a suitable species to study dysregulated glutamine metabolism in aged HSCs. Together this work demonstrates that aging causes a disruption of glutamine and glutamate metabolism in HSCs. The cause for this disruption and its direct consequences on overall HSC metabolism remains to be explored. However, it is likely that these metabolic changes will have a knock-on effect on HSC function and may elucidate some of the drivers of metabolic HSC aging.

MiR-9-1 Controls Osteoblastic Regulation of Lymphopoiesis

The highly conserved MicroRNA-9 (miR-9) family consists of three members, miR-9-1, miR-9-2 and miR-9-3. These three microRNAs are encoded by unique genes located on different chromosomes, the transcripts of which are processed into the same mature miR-9 sequence. MiR-9 is known to play a crucial role in brain development and neurogenesis, its involvement in hematopoiesis has remained uncertain. To explore this, we investigated the role of miR-9-1 in controlling hematopoiesis. Our findings revealed that miR-9-1 and miR-9-2 but not miR-9-3 were expressed in all hematopoietic cells, with higher levels observed in HSCs, MPPs, and CLPs. In order to understand the impact of miR-9 on hematopoiesis, we employed CRISPR/Cas9-mediated genome editing to create miR-9-1-deficient mice. Deletion of miR-9-1 resulted in reduced mature miR-9 expression, leading to 43% of the mice exhibiting retarded growth and postweaning lethality by 12 weeks of age. The growth-retarded miR-9-1-deficient mice experienced severe lymphopenia, primarily affecting B and T cell development. Further analysis revealed that the populations of pro/pre- and immature B cells in bone marrow (BM) were dramatically reduced in growth-retarded miR-9-1-deficient relative to wild-type control mice. The populations of peripheral T1, T2, FO and MZ B cells were also markedly reduced in growth-retarded miR-9-1-deficient relative to control mice. In addition, the numbers of DN, DP, and SP thymocytes and CD4 + and CD8 + T cells in the spleen were also markedly reduced in growth-retarded miR-9-1-deficient relative to control mice. Consistent with the specific impairment of lymphocyte (B and T cell) development and maturation, the growth-retarded miR-9-1-deficient mice exhibited a reduction of hematopoietic progenitors including ST-HSCs, MPPs, CMPs and especially CLPs, but had normal numbers of GMPs and MEPs. Interestingly, transplantation of miR-9-1-deficient BM cells into wild-type recipients resulted in normal lymphopoiesis, indicating that the lymphopenia was not due to hematopoietic cell autonomous effects but was caused by alterations in the BM stromal microenvironment. We then focused on the effect of miR-9-1 deficiency on the BM niche, particularly the mesenchymal stem cell (MSC) and osteoblastic (OB) niches. The numbers of MSCs in BM stromal cells were comparable between miR-9-1-deficient and wild type mice, whereas the number of osteoblastic cells (OBs) was significantly reduced in the BM stromal cells from growth-retarded miR-9-1-deficient relative to wild-type mice. In agreement with the reduction of OBs in miR-9-1-deficient mice, the mutant MSCs failed to differentiate into OBs. Notably, miR-9-1 deficiency did not influence the differentiation of osteoclasts and adipocytes, suggesting a specific impairment in osteoblastic bone formation. To elucidate the molecular mechanism underlying miR-9-1's regulation of osteoblastic differentiation, we conducted high-throughput RNA sequencing analysis on miR-9-1-deficient MSCs and OBs. The results revealed reduced expression of master transcription factors involved in osteoblastic differentiation, Runt-related transcription factor 2 (Runx2) and Osterix (Osx), and genes related to collagen formation, extracellular matrix organization, and cell adhesion, in miR-9-1-deficient MSCs. Additionally, we identified Follistatin (Fst) as a direct target of miR-9-1. MiR-9-1 deficiency led to increased Fst levels, which, in turn, inhibited BMP signaling in MSCs and reduced IL-7 and IGF-1. In summary, our studies demonstrate that miR-9-1 plays a pivotal role in controlling osteoblastic regulation of lymphopoiesis by targeting the Fst/BMP/Smad signaling axis.

Combined Single-Cell and Spatial Transcriptomics Unveil the Complex Organization of the Non-Immune Human Bone Marrow Microenvironment during Aging

Hematopoietic stem cells (HSCs), reside in a specialized bone marrow (BM) microenvironment known as the hematopoietic stem niche. Despite the critical role of the niche in tightly controlling the processes of normal and malignant hematopoiesis, its cellular composition remains only partially understood. Recent studies, including our own, using single-cell RNA sequencing (scRNA-seq) approaches have provided valuable insights into the profiling of the human BM niche. However, a comprehensive effort to describe the cellular heterogeneity and regulatory circuitry of the aged BM microenvironment in elderly humans is still lacking. As individual age, multiple systems and organs experience a progressive loss of anatomical and physiological integrity. Aging of the HSC niche is accompanied by a reduction in the numbers and function of its constituents and a decrease in the levels of HSC-supporting factors. Whether niche aging can contribute to the defects observed in aged hematopoiesis, such as the clonal shift towards myelopoiesis, the decrease in immune surveillance, or age-associated metabolic diseases, remains unresolved. To address this, we utilized scRNA-seq and spatial transcriptomics to provide a detailed characterization of the molecular landscape and stromal interactions in the aged non-immune BM microenvironment. First, we performed scRNA-seq profiling on fluorescence-activated cell sorting-purified endothelial cells (ECs, TO-PRO-3 -/CD45 -/CD235 -/Lin -/CD31 +/CD9 +) and mesenchymal stromal cells (MSCs, TO-PRO-3 -/CD45 -/CD235 -/Lin -/CD271 +) from human BM samples of young (n=4) and elderly healthy donors (n=5). We analyzed a total of 1514 ECs and 3848 MSCs from older adults, grouped into 7 and 10 subclusters, respectively, defining distinct functional cell states. ECs and MSCs cells from young individuals were annotated using SingleR, utilizing the gene signature of each functional state in the elderly as a reference. Our results revealed significant shifts in the distribution of the functional states of BM niche cells during aging. Regarding ECs, we noticed reduced pathways associated with the cell cycle and RNA transcription pathways, indicating impaired cell cycle activity, coupled with decreased antioxidant defense (Figure 1A). In contrast, there was an increase in subgroups related to the response to foreign molecules, immune system activation, and vascular remodeling, suggesting an inflammatory response and increased vascular remodeling processes associated with aging. For MSCs, we observed a decline in osteogenesis and a reduction in the proportion of the early mesenchymal group related to immunity and the extracellular matrix (Figure 1B). These changes could contribute to the decreased ability of MSCs in elderly individuals to maintain HSCs function. On the contrary, we noticed increased adipogenic differentiation to the detriment of osteogenesis, which could be responsible for osteoporosis problems. Next, and to further explore the location and the direct interactions between niche cells, we are currently integrating spatially resolved transcriptomics with our scRNA-seq data to accurately comprehend the spatial distribution and interactions among the BM niche cells, using for the first time 10x Genomics Visium Spatial Gene Expression on bone BM tissue in humans. As a final validation step, we are using a mouse model to validate further the mechanisms of age-associated changes in the BM microenvironment. In summary, our results provide valuable insights into age-related transcriptional alterations in human BM ECs and MSCs, suggesting altered behavior of these niche cells during the aging of the hematopoietic system. This deeper understanding of the architecture of the aged hematopoietic system and its microenvironment offers the potential for developing novel therapeutic strategies preventing the detrimental effects of aging.

Tuning the Innate Immune Multiple Myeloma Microenvironment By Modulating IRF4

The tumor microenvironment (TME) has emerged as a key contributor to cancer progression in numerous solid tumors as well as blood cancers that evolve in the bone marrow (BM). In the context of multiple myeloma (MM), survival of malignant plasma cells is supported by interactions with the BM microenvironment, which is comprised of various components including cells, extracellular matrix, and soluble factors. Therefore, the development of strategies that specifically target the MM BM microenvironment is crucial for improving current disease prognosis. Within the BM niche, M2-like myeloma-associated macrophages (MAMs) play a significant role in cancer cell survival and progression and have been implicated in producing an immune-suppressive TME by generating inflammatory mediators, growth factors, cytokines, chemokines, etc. Macrophages exhibit plasticity with regard to their polarization state, which enable them to dynamically respond and adapt to different environmental stimuli in order to fulfill important immune functions. Thus, the reprogramming of M2-like, pro-tumoral MAMs toward a M1-like, anti-tumoral phenotype represents a promising therapeutic strategy. In MM, inflammatory and anti-viral pathways promote disease progression. Interferon regulatory factor 4 (IRF4) is a transcription factor that has previously been shown to promote MM progression through enhanced survival of malignant plasma cells. Under physiological conditions, IRF4 also regulates macrophage polarization, where its expression drives alternative activation of macrophages primarily to M2-like (anti-inflammatory) phenotypes. However, the extent to which IRF4 activation or direct inhibition governs the polarization status and pro-tumoral activity of macrophages in the MM microenvironment has not been fully elucidated. In this study, we sought to further explore the role of IRF4 in macrophage polarization and investigated the potential of modulating IRF4 expression to reprogram myeloma-associated macrophages (MAMs) as a novel therapeutic strategy. First, we developed myeloma-tailored macrophage polarization models using primary human peripheral blood-derived monocytes and the THP1 monocyte cell line. Our M1 and M2 polarization models utilize a customized cocktail of myeloid growth factors and cytokines, supplemented with interleukin (IL)-6 to mimic the IL-6-enriched MM microenvironment. Characterization of M1 (CD80, TNFα, and CXCL10) and M2 (MRC1, FN1, and CCL22) markers confirmed the presence of distinct macrophage subpopulations, as well as upregulated expression of IRF4 in M2-like progeny and IRF5 in M1-like macrophages. To investigate the extent to which IRF4 activation or direct inhibition governs macrophage polarization and function, overexpression and knockdown experiments on macrophages were performed in vitro, along with whole transcriptome sequencing of IRF4-overexpressing THP1 monocytes compared to vector-transduced controls. Gene expression analyses revealed that genetic down-modulation of IRF4 in M2-like macrophages shifts the polarization status toward a hybrid phenotype that displays enhanced M1-like properties, while still retaining M2-like characteristics. This shift in polarization state was further amplified when monocytes were transduced to overexpress IRF4 prior to differentiation into M2-like macrophages and subsequent IRF4 knockdown, suggesting that IRF4 plays a key regulatory role in tuning macrophage polarization. Taken together, our preclinical findings provide groundwork for future studies on MAMs and suggest that an IRF4-mediated macrophage reprogramming strategy is a promising therapeutic approach for patients with MM and a variety of other malignancies typified by an anti-inflammatory TME.

Instructive Interaction between Myelodysplastic Hematopoiesis and the Bone Marrow Microenvironment at Single Cell Level

Myelodysplastic Neoplasms (MDS) are hypothesized to re-model their bone marrow (BM) microenvironment to re-inforce conditions for their propagation. In this study, we investigated interactions between MDS cells and the BM niche at single cell level in a MDS patient-derived xenograft model (PDX) as well as primary human MDS and healthy (HY) bone marrow (BM samples in order to identify cues of disturbed crosstalk between MDS hematopoiesis and the BM microenvironment. In the PDX model, we analyzed >13,000 cells of n=15 different murine niche cell populations after long-term (>24 weeks) exposure to n=3 MDS patient samples versus n=2 age-matched HY human grafts. Subsequently, we analyzed over 24,000 primary human BM cells enriched for the non-hematopoietic compartment by using whole bone fragments from n=8 MDS patients and n=7 HY age-matched donors. All cells were analyzed with combined 10x single cell RNA sequencing and human “Cellular Indexing of the Transcriptome and Epitopes by Sequencing” (CITE Seq). Results were validated by flow cytometry and gene expression analyses. Mesenchymal cell (MSC) subpopulations from PDX mice exposed to MDS versus HY hematopoiesis could be subdivided into distinct “Cxcl12+ abundant reticular” (CAR) cells with osteogenic and adipogenic differentiation trajectories. Various CAR cell populations displayed an increased frequency in MDS transplanted mice as compared to PDX transplanted with HY grafts ( Panel A). Upon contact with hematopoietic MDS cells, CAR cell populations overexpressed numerous genes involved in extracellular matrix reorganization and hematopoietic supporting factors, such as Cxcl12, Il7, Lox and Loxl1 as compared to mice bearing HY grafts. In situ, BM cells from MDS patients showed highly heterogeneous MSC subpopulations with similar differentiation trajectories to the ones observed in our PDX models on a patient-individual level. We detected two non-hematopoietic cell populations, designated as endothelial cells (ECs) and MSCs characterized by expression of CXCL12, IGFBP5 or COL1A2 and quantification with epitope sequencing (CITE Seq) with mutually exclusive detection of CD31 and CD271 expression (n=581 cells in total, Panel B). In n=3 MDS patients, we identified an MDS-specific MSC subpopulation with high IGFPB4 and CXCL12 expression as well as overexpression of KITLG and IL7. This suggests that this altered specialized secretory mesenchymal state is present in a subset of MDS patients and may support the malignant hematopoietic clones by overexpression of key hematopoietic factors. Interactome analyses using spatial receptor:ligand bioinformatic tools such as CellphoneDB, RNAMagnet and omnipath further confirmed that stromal cells alter their cellular profile and secretory behavior in response to MDS hematopoiesis. In conclusion, we show that murine BM stroma is re-programmed by exposure to human MDS xenografts to upregulate extracellular matrix (ECM) re-modeling and HSC supporting factors. In native human MDS BM in vivo, rare primary mesenchymal cells are re-programmed to increase hematopoietic support in a patient-individual manner pointing to altered MSC subpopulations with increased growth factor expression profiles in a subgroup of MDS patients.

Efficient and Specific In Vivo Genetic Engineering of Human Hematopoietic Stem Progenitor Cells without Selective Conditioning

Achieving in vivo genetic engineering of hematopoietic stem progenitor cells (HSPC) has the potential to transform treatment for hematological disorders. An ideal in vivo delivery platform should provide access to resting HSPC at low doses, in different in vivo compartments and with high specificity. We developed a multi-model strategy to assess efficiency and specificity of access to human HSPC, without reliance on conditioning to selectively enrich for gene-modified cells, and we achieved efficient genetic engineering of Lin-CD34+ cells and individual HSPC subtypes using highly potent lentiviral vectors (LV) and gene editing virus-like particles (VLP). We first conducted a high-resolution characterization of access to resting HSPC and individual FACS-sorted HSPC subtypes in vitro with different LV envelopes, including a detailed analysis of insertion site distribution in resting vs cytokine activated CD34+ cells. With a BaEVTR LV we achieved transduction of ~100% resting HSPC at MOI 66 (vs ~40% with a VSV-G LV at MOI 50,750) and ~20% of FACS-sorted hematopoietic stem cells (HSC) at MOI 32. This translated into 2-4% transduction of bone marrow (BM) huCD45+ cells when the same BaEVTR LV was dosed in NBSGW long-term humanized mice. However, such models carry a highly lymphoid-skewed human hematopoiesis and are not well suited for measuring access to all HSPC subtypes in all compartments. We therefore established 2 additional in vivo experimental scenarios. To measure LV access to HSPC in the peripheral blood (PB), we followed long-term NBSGW mice where BaEVTR LV was dosed intravenously (IV) immediately after HSPC infusion. In this model we achieved targeting of 23% early engrafting BM HSPC which was mirrored by a ~20% transduction in PB myeloid cells during the first 6 weeks, later stabilizing at 5-7% from week 8 up to week 16. To then measure access to HSPC in the BM niche, we characterized early human HSPC dynamics in the BM of NBSGW mice along 5 timepoints over the first 19 days after CD34+ cell infusion, with or without AMD3100/G-CSF mobilization. We then dosed BaEVTR LV at D7 post-humanization, a timepoint when we established that engrafted human cells have a more physiological lineage distribution, are few and remained confined in the BM even upon mobilization. In this model we demonstrated that a single BaEVTR LV dose injected IV can reach 5-6.4% of BM-resident HSPC. Based on these data, we developed a BaEVTR VLP carrying a CRISPR/Cas9 editor targeting the B2M locus. This VLP displayed 93% editing efficiency in vitro in resting HSPC and 38 to 61% in FACS-sorted HSC at MOI 16 and MOI 83 respectively. When dosed in vivo in the D7 post-humanization model, our VLP achieved B2M KO in 16% and 31% BM-resident Lin-CD34+ cells with 1 and 3 consecutive IV injections respectively, including 14% of phenotypic HSC. Importantly, a comparable level of in vivo editing of BM Lin-CD34+ cells (12%) was achieved upon equivalent pilot single VLP dosing in long-term humanized mice despite them carrying substantially higher numbers of human HSPC in the BM and a complete and stable PB human blood cell engraftment. Secondary transplantation experiments to confirm editing of long-term HSC are ongoing. We proved delivery of genetic payloads to resting HSPC in vitro and access to HSPC in different compartments in vivo with high efficiency. Adding a specific tropism for HSPC has the potential to enhance on-target bioavailability impacting effectiveness and safety of our delivery systems. To this aim, we are now testing LV and VLP carrying HSPC-targeted envelopes engineered with viral fusion proteins (retargetable fusogens). We will present comprehensivedata showing that a LV pseudotyped with a CD133-targeted fusogen combines in vitro and in vivo potency on par with BaEVTR LV with high specificity for CD133+ cells, as seen upon titrations on 8 cell lines and 10 primary human cell types as well as in spike-in experiments aimed at measuring CD133+ HSPC transduction when diluted in a mixture of CD133- human blood cells in vitro and in NBSGW mice. Furthermore, we will show that a CD117-targeted LV can transduce, in vivo, a cell population exclusively located in the BM and comprising as little as 0.3% of total huCD45+ cells with a 175-fold increased specificity as compared to envelopes with broad specificity. These results set the basis for generating a potent candidate for in vivo delivery of genetic payloads to HSPC with high efficiency and specificity.

A c- MPL Splice Variant up-Regulated in Myeloproliferative Neoplasms Modulates Thrombopoietin Signaling and Induces Cycling of Hematopoietic Stem Cells and Inflammatory Progenitors

Introduction: Myeloproliferative neoplasms (MPN) are clonal disorders caused by mutations that activate JAK/STAT signaling through the thrombopoietin receptor, MPL, leading to overproduction of myeloid lineages. MPL is the only hematopoietic growth factor receptor expressed on the surface of hematopoietic stem cells (HSC) where it maintains their quiescence by tethering HSCs to osteoblasts in the endosteal niche. MPL is also expressed on megakaryocytes and platelets. Individuals born with loss of function mutations in the distal MPL cytokine receptor homology domain (CRHD) develop thrombocytopenia and progressive bone marrow failure. How MPL functions in MPN remains unclear. Here, we describe a novel MPL splice variant ( MPL-SV) that is present in CD34 + cells and platelets from healthy controls, but overexpressed in the MPN. We therefore sought to: 1) define MPL-SV function in hematopoiesis and MPN, and, 2) elucidate mechanisms underlying MPL-SV. Methods: After identifying a novel MPL-SV transcript from JAK2 V617F MPNplatelets, we compared its expression to MPL transcripts lacking the deletion in MPN patients and healthy controls. To assess its function, we generated transgenic (Tg) mice expressing varied levels of MPL-SV in HSC. Next, we examined responses of the Tg mice to thrombopoietin (THPO), hematopoietic stem progenitor cell (HSPC) frequency by flow cytometry, and HSPC function by colony-forming assays (CFA). To elucidate mechanisms, we performed single cell RNA sequencing (scRNAseq) of bone marrow (BM)-derived Lin -, Sca +, c-kit + (LSK) cells followed by pathway analysis. Results: Using primers to amplify the MPL CRHD in platelets from patients with JAK2 V617F MPN, we discovered an MPL- SV lacking 7 amino acids in the MPL amino-terminal domain, a common hotspot for activating MPN mutations. MPL-SV is present in CD34 + cells and platelets of healthy controls, but overexpressed in MPN relative to MPL transcripts lacking the deletion. To examine its function, we generated transgenic (Tg) mice expressing MPL-SV in HSC. Tg miceexpressing a single copy of MPL-SV have unperturbed hematopoiesis, whereas Tg mice expressing 6 copies of the transgene ( MPL-SV-6) develop marked thrombocytopenia, mild splenomegaly, and a modest increase in red cells by 9-12 weeks of age. Baseline serum THPO levels are elevated in Tg MPL-SV-6 mice and they fail to increase platelet counts following exogenous THPO administration, suggesting defective function of MPL-SV in binding and/or internalizing THPO. To further assess MPL-SV function, we compared CFA from HSPC of Tg MPL-SV-6 and wildtype mice, which show a decrease in total colony forming units (CFU) from BM or spleen-derived MPL-SV-6 HSPC. Accordingly, there was a decrease in LSK cells, multi-potent progenitors (MPPs), and megakaryocytes (Mk) in BM by flow cytometry. By contrast, spleens show increases in LK cells and short-term HSC. Further analysis by scRNAseq of BM-derived LSK cells reveal a marked decrease in both quiescent HSC and HSC with a Mk-bias associated with a relative expansion in more proliferative HSC, including those with a bias towards erythroid and mature myeloid differentiation. Pathway analysis reveal that MPL-SV activates transcriptional networks involved in TNF-α signaling via NF-κB and oxidative phosphorylation. Analysis of cell-cell interactions by CellChat show decreased signaling through intercellular adhesion molecule 1 (ICAM-1) in MPL-SV-6 HSC compared to controls. Importantly, ICAM1 maintains quiescent HSC within their BM niche. Similarly, cell cycle analysis from scRNAseq indicate that quiescent HSC are stimulated to cycle and generate HSC primed to differentiate into erythroid and myeloid progeny. Conclusions: We discovered a novel MPL-SV that is up-regulated in MPN. In Tg mice, MPL-SV impairs responses to THPO and disrupts hematopoiesis by depleting quiescent HSC and BM megakaryocytes. Single cell transcriptomics suggest that MPL-SV drives expansion of cycling HSC and HSC primed to differentiate into progenitors involved in inflammation, including myeloid cells. Mechanistically, HSPC expressing MPL-SV-6 activate transcriptional networks involved in TNFα signaling via NF-κB pathways. Our results highlight the central role for MPL in hematopoiesis and suggest that aberrant MPL splicing contributes to MPN pathogenesis.

Pharmacology of a CpG-Linked Anti-Mir-126 Oligonucleotide Targeting Acute Myeloid Leukemia (AML) in Rats and Non-Human Primates (NHP)

Introduction: Acute myeloid leukemia (AML) is a lethal hematopoietic malignancy, characterized by the accumulation of clonal myeloid progenitor cells arrested in development. AML patients have dismal outcomes with a 5-year overall survival (2011 - 2017) of 29.5% (SEER). The persistence of leukemia stem cells (LSCs), a treatment-resistant primitive cell population able to inititiate and maintain AML, is thought to underlie relapse. Even newly approved drugs do not target LSCs efficiently. MicroRNA (miR)-126 is a non-coding regulatory RNA that contributes to LSC homeostasis in the bone marrow niche. Several lines of evidence validate miR-126 as a target in AML. Thus, we designed and made a novel anti-miR-126 oligonucleotide (oligo) linked to a CpG moiety, termed miRisten. miRisten incorporates modifications such as 2'-fluoro and phosphorothioate substituting phosphodiester in the sugar moiety of the nucleotide to enhance in vivo stability. miRisten eradicates LSCs in mouse models of AML and synergizes with FDA-approved AML drugs (PMID: 29505034, 34372909). Here, the results of IND-enabling toxicokinetic (TK) studies of miRisten in rats and NHP are presented. Objective: To investigate the pharmacological properties of miRisten to establish a safe dosing strategy to achieve prolonged knockdown of miR-126 for a first-in-human trial. Experimental Plan and Results: Good laboratory practice (GLP) TK studies were carried out in Sprague-Dawley rats. miRisten was given IV b.i.d. for 19 days at human equivalent doses (HED) of 1.6 and 4 mg/kg. miRisten was measured in plasma after the first dose. Plasma miRisten C max average was 3622 ± 335 and 6010 ± 175 nM post first doses of 1.6 and 4mg/kg, respectively. Plasma t 1/2 averaged 20.0 ± 3.8 minutes across both doses. The area under the curve (AUC 0 - ¥) were 638.9 ± 32 nMxhr (1.6 mg/kg) and 1313.5 ± 439 (4 mg/kg). A parallel toxicology study was undertaken documenting cage-side observations and body weight; complement activation, blood chemistries, urine and hematology were also evaluated. There were no unscheduled deaths in the toxicology segment. Four single NHP exploratory studies were also carried out (Fig. 1: Treatment schema). In each study, miRisten was given IV as a slow bolus (~5') b.i.d. and PK parameters evaluated in plasma. Study #1 & #2: 1 wk at 4mg/kg; #3: 1 wk at 5 mg/kg; #4: 18 days at 4 mg/kg. In all studies, miRisten dosage was ramped up (1, 2, 4 mg/kg for all and 5 mg/kg at dose 4 for Study #3). Solumedrol was given as premedication for the first 3 doses in all studies to prevent known phosphorothioate-mediated complement activation and cytokine release syndrome. Blood draws for PK were carried out immediately following doses 3 and 12 (Studies 1 - 3) and after the last dose on Day 18 (Study 4). The average area under the curve AUC was 3860 ± 1189 (4 mg/kg) and 3954 ± 1569 nMxhr (5 mg/kg) which was greater than observed in mice given the equivalent HED in bolus doses (~6x, 465.0). The t 1/2 of plasma miRisten was 11.7 min on Day 3 and 12.0 min on Day 7, comparable to mouse (9.6 min) and rat (20 min). Cage-side observations, body weight, complement activation, blood chemistries, urine and hematology were also evaluated. There was no terminal sacrifice of NHP however, all animals tolerated the treatment without observable adverse events. Conclusion: The dose ramp-up in NHP combined with pre-medication with solumedrol were undertaken to prevent possible acute inflammatory reactivity leading potentially to death. There were no unscheduled deaths in any species studied. These data are consistent with the safety of miRisten given IV with a dose ramping regimen combined with solumedrol. The greater AUC in NHP may predict better potency in humans. In mice, miRisten at 1.6 mg/kg HED q.d. effectively eradicated LSCs [PMID 29505034].

NFκB Dysregulation in the Bone Marrow Niche Drives Epigenomic Reprogramming of Hematopoietic Stem Cells and Myeloid Bias

Introduction Aging and chronic inflammation are known to skew hematopoietic output by favoring myelopoiesis and disfavoring lymphopoiesis. In addition, the bone marrow niche plays critical roles in supporting steady-state hematopoiesis, and alterations in the microenvironment may similarly influence disordered hematopoiesis. The NFkB system is a central, pleiotropic regulator of inflammatory signaling in many cell types. Thus, we reasoned that experimental perturbations of NFκB signaling may be used to study how inflammatory dysregulation affects hematopoiesis. Methods We generated a mouse model of inflammatory immune dysregulation, the IκB - mouse ( Nfkbia +/- Nfkbib -/-Nfkbie -/-), and then compared the peripheral blood and bone marrow inflammatory proteins and cell compositions betweenyoung WT, young IκB -,and aged WT mice. Using bone marrow transplantation, we next assayed the effect of an inflamed IκB - recipient niche on the hematopoietic output of WT donor bone marrow. Finally, we performed single cell RNA sequencing (scRNA-seq) and assay for transposase-accessible chromatin sequencing (ATAC-seq) on sorted hematopoietic stem and progenitor cells (HSPC) to characterize the epigenomic and transcriptional changes occurring in WT HSPC from an immune dysregulated environment. Results We observed an increase in inflammatory cytokines in both young IκB - and aged WT mice compared to young WT controls, as well as myeloid bias in the peripheral blood leukocytes and bone marrow HPSC. Transplantation studies showed that inflammatory dysregulation in the radio-resistant microenvironment resulted in myeloid-biased hematopoiesis. However, this myeloid bias was lost upon secondary transplantation of bone marrow into a WT recipient. Performed on HSPC from an inflamed microenvironment, our scRNA-seq studies showed myeloid bias at the transcriptional level, and ATAC-seq studies further revealed epigenomic changes, with an increased transcription factor (TF) binding motif accessibility for myeloid-priming TFs in long term HSC. Conclusions Our studies establish the IκB - mouse as a new model of chronic inflammatory dysregulation that can be used to study the effects of an inflamed bone marrow microenvironment on hematopoiesis. Furthermore, our findings indicate that dysregulated NFκB signaling in the bone marrow microenvironment leads to myeloid bias in HSPC, and is associated with epigenomic reprogramming at the stem cell level. However, despite this epigenomic reprogramming, we observed that myeloid bias could be reversed upon bone marrow re-transplant into a non-dysregulated niche. Together, these data suggest that further understanding niche-derived inflammatory dysregulation may be a useful in targeting disordered hematopoiesis in chronic inflammatory states such as aging.

Epigenetic Scarring Leads to Irreversible NK Cell Dysfunction in Myeloid Malignancies

Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) belong to the same spectrum of myeloid malignancies and have extremely poor outcomes in the relapsed/refractory setting. Despite advances in the understanding of the pathogenesis and molecular mechanisms of these disorders, and incremental improvements in treatment regimens, patients with MDS and AML often relapse and fail to achieve cure. These and other factors underscore the urgent need for new therapeutic alternatives that will improve the clinical outcomes of these patients. Immunotherapy using checkpoint molecule inhibitors and adoptive cell therapy using autologous immune effector cells have been mostly unsuccessful in patients with MDS and AML. This could be due to the immunosuppressive tumor microenvironment in the bone marrow niche or to intrinsic dysfunction in the immune effector cells of these patients. Natural killer (NK) cells are innate lymphocytes that play an important role in cancer immune surveillance and have distinct advantages over T cells as candidates for immunotherapy. Myeloid blasts are inherently susceptible to NK cell-mediated killing as they express many of the ligands recognized by NK cell activating receptors. However, malignant myeloid blasts are capable of adapting and developing defense mechanisms that allow them to evade NK cell-mediated cytotoxicity. Little is known about the mechanisms of NK cell immune evasion developed by myeloid blasts. Here, we show that NK cells from patients with myeloid malignancies display a global dysfunction with severely impaired secretory function and killing capacity. Single cell RNAseq and mass cytometry experiments revealed that these NK cells from MDS and AML patients display an exhausted phenotype at the transcriptomic and proteomic levels. We also show that these NK cells have an altered metabolism compared with age matched healthy control NK cells. We show that this dysfunction is mediated by a crosstalk between myeloid blasts and NK cells and cell-cell contact dependent release of transforming growth factor beta (TGF-β). This crosstalk leads to a profound epigenetic reprogramming of NK cells driven by transcription factors known to mediate exhaustion and immune suppression. Myeloid blast-induced NK dysfunction and epigenetic state of exhaustion can be prevented by pharmacologically inhibiting the TGF-β pathway or knockout of TGFBR2 in NK cells. However, our data reveal that once this dysfunction occurs it is irreversible owing to epigenetic scarring driven by the transcription factor BATF. In fact, we show that TGF-β induces the expression of BATF in NK cells, which in turn mediates a gene regulatory program leading to NK cell dysfunction by driving the expression of exhaustion and inhibitory genes (e.g. HAVCR2, ENTPD1, CTLA4, TGFBR2). Collectively, our findings reveal a novel mechanism of NK cell immune evasion manifested by stable epigenetic rewiring and inactivation of NK cells by myeloid blasts. Our data support the use of allogeneic sources for adoptive NK cell therapy in combination with strategies aiming at preventing immune suppression to treat myeloid malignancies rather than therapies aiming at reversing or rescuing the function of autologous NK cells.

The ATF4-RPS19BP1 Axis Modulates Ribosome Biogenesis to Promote Erythropoiesis

The function of hematopoietic stem cells (HSCs) and the process of hematopoiesis are intricately governed by a complex interplay of intrinsic programs and extrinsic signals from the microenvironment (Crane et al., Nat Rev Immunol, 2017). Previous work reported by our group and others has demonstrated that ATF4, a basic region-leucine zipper transcription factor, plays pivotal roles in fetal liver hematopoiesis, HSC maintenance, bone formation and tumorigenesis (Zhao et al., Blood, 2015; Sun et al., Sci Adv, 2021; Yang et al., Cell, 2004). However, the precise function of ATF4 in the bone marrow niche and how ATF4 regulates adult hematopoiesis remain largely unknown. Here, we employ four cell-type-specific mouse Cre lines to conditionally knock out Atf4 in Cdh5 + endothelial cells (ECs), Prx1 + bone marrow stromal cells (BMSCs), Osx + osteo-progenitor cells, and Mx1 + hematopoietic cells, and uncover the role of Atf4 in niche cells and hematopoiesis. We found that Atf4 deletion in BMSCs reduced BMSC numbers and impaired their CFU-F activity as well as differentiation toward osteoblasts, but spares hematopoiesis and HSC function in adult mice. Furthermore, Atf4 depletion in the ECs or osteo-progenitors of adult mice has a very mild effect on the BM hematopoiesis and HSC maintenance. Intriguingly, Atf4 depletion in the hematopoietic system resulted in severe anemia. More than 70% of the Mx1cre; Atf4 fl/fl (Δ/Δ) mice gradually died from anemia. Atf4 deficiency markedly reduced the frequency of erythroid progenitor cells (EPCs) (including MEP, PreCFU-E, and CFU-E cells) and impaired their CFU-E and BFU-E colony-forming ability. Atf4 depletion severely impaired the repopulation ability of BM cells and the self-renewal capacity of HSCs. Single cell RNA-seq (scRNA-seq) analysis of BM Lin -cKit + cells confirmed the defects of erythropoiesis after Atf4 depletion. The absence of ATF4 compelled early erythroid progenitors to enter the S phase, which led to replication stress and in turn activated both the DNA damage response and apoptosis. Subsequently, we conducted an integrative analysis of the genes that were identified as downregulated in MEPs by RNA-seq, ATAC-seq, and H3K4me3 Cut&Tag and genes that were identified as downregulated in erythroid progenitors (i.e., PreMegE, Ery1, and Ery2) by scRNA-seq, which predicted Rps19bp1 as the top target gene of Atf4. Luciferase reporter and quantitative chromatin immunoprecipitation (qChIP) assays confirmed that ATF4 was a transcriptional activator of Rps19bp1. Downregulation of Rps19bp1 caused by Atf4 deletion led to decreased assembles of 40S proteins, such as RPS3, RPS6 and RPS19. O-propargyl-puromycin (OP-Puro) incorporation and SUnSET assay further confirmed the global protein synthesis defect in Atf4-deficient MEP cells. The protein synthesis defects and impaired erythropoiesis were rescued upon RPS19BP1 overexpression in Atf4 knockdown MEL cell line or BM cKit + cells from Atf4-deficient mice. Ribosome profiling of CMP cells from Δ/Δ and fl/fl mice further demonstrated that the expression of proteins involved in ribosome biogenesis was significantly lower in Atf4-depleted than in wildtype CMP cells. The expression of gene sets linked to PreCFU-E signature was also lower in Atf4-depleted than in wildtype CMPs, suggesting that the reduction in ribosome biogenesis significantly affected the translation efficiency of erythroid-related pathway components, thereby impeding erythroid lineage commitment. Finally, we demonstrate that under conditions of 5-fluorouracil-induced stress, Atf4 depletion impedes the recovery of hematopoietic lineages, which requires efficient ribosome biogenesis. Taken together, we demonstrated that, unlike in the fetal liver, ATF4 governs adult HSC function and erythropoiesis in a cell-intrinsic manner. We revealed a novel role for ATF4 in erythropoiesis, which links it to RPS19BP1, ribosome biogenesis and protein translation. Our findings have highlighted the crucial importance of protein synthesis regulation during erythroid lineage commitment. This discovery will likely have extended implications for understanding and treating ribosomopathy-associated erythroid failure.

Inflammation Shapes the Transcriptional Landscape of the Bone Marrow Microenvironment in Post-Transplantation Leukemia Relapses

Allogeneic Hematopoietic Cell Transplantation (allo-HCT) represents the most successful therapeutic option for many patients suffering from Acute Myeloid Leukemia (AML). Nevertheless, leukemic cells often find means to evade control from the donor-derived immune system and re-emerge. To date, three alternative and frequent mechanisms by which AML cells modify their features and escape immune control have been described: genetic loss of the mismatched HLA haplotype (HLA loss), downregulation of HLA class II molecules and upregulation of inhibitory ligands, all ultimately leading to abolished leukemia recognition by T lymphocytes. However, increasing evidence suggests that leukemic cells weave a much broader net of interactions, involving several other immune cell types within the bone marrow microenvironment. Still, how this complex crosstalk between malignant cells and the bone marrow niche may shape the features of leukemia post-transplant relapse remains unknown. By exploiting the high resolution provided by single-cell RNA-sequencing (scRNA-seq), we provide a detailed insight of the human bone marrow immune microenvironment and the changes that occur following allo-HCT, both when disease reemergence is prevented, and when it occurs. Using cryopreserved BM aspirates, we profiled a total of 145,420 mononuclear cells from 25 adult AML patients at the time of disease relapse; 56,831 cells from 5 post-transplant patients in complete remission (CR), each tested at two different timepoints (+90 and +365 days) and 45,279 cells from 6 healthy controls (HC). First, scRNA-seq allowed us to recapitulate known leukemia-intrinsic features of each relapse modality, such as HLA loss, down-regulation of HLA class II molecules and upregulation of inhibitory T cell ligands. Moreover, combining genotype and Copy Number Variation (CNV) inference algorithms, we were able to separate malignant cells from their healthy counterparts. Interestingly, HLA loss relapses exhibited a more immature profile, with a significantly higher leukemia stem cell signature (LSC17) (p-value < 0.0001, Wilcoxon rank-sum test) (Ng SW et al., Nature 2016), hinting that the hematopoietic cell of origin impacts on the mechanism of post-transplantation relapse. Leukemic cells also featured a high inflammation-associated gene score (iScore) (Lasry A et al., Nat Cancer 2023), as also evident in non-malignant HSPCs from relapsed patients, which showed an increased IFN-α signature compared to HC and CR. However, AML samples that couldn't be categorized by a known mechanism of relapse displayed a significantly lower iScore, suggesting that leukemia escape in these patients might not have had an immune-related driver (Figure 1a). We next sought to characterize specific BM immune compartments. We overall detected a lower frequency of CD4 T cells in relapsed patients as compared to HC and CR patients (p-value = 0.04, Wilcoxon rank-sum test). We annotated 5 major Natural Killer (NK) cell clusters (immature CD56 dim, mature CD56 dim, CD56 bright, proliferating NK, NKT cells) and a smaller subset of immature CD56 bright NK cells characterized by higher expression of IFN-related genes and NK exhaustion markers, which was defined as inflamed NK cluster. Inflamed NK cells were more represented in patients with upregulation of T cell inhibitory ligands compared to other relapsed patients (5.42% of total NK vs. 0.74%, respectively. p-value = 0.03, Wilcoxon rank-sum test), thus suggesting a shared mechanism of dysfunction signature between T and NK cell that may favor leukemia immune evasion (Figure 1b). In conclusion, we generated a comprehensive atlas of the BM immune microenvironment in AML post-transplantation relapses, showing that inflammation shapes immune subset composition and transcriptional profile in the BM, and that leukemia mechanisms of immune evasion are influenced by the surrounding milieu. Overall, while an in depth understanding of whether leukemia relapse is caused by an inflamed microenvironment, or vice versa, might only be achieved through longitudinal sampling from patients before and after transplant, our results here exemplify how the BM niche could be exploited to find novel vulnerabilities in AML relapses, to be specifically targeted by personalized therapeutic approaches.

Detection and Prevention of ADAR1p150-Induced Hematopoietic Stem and Progenitor Cell Aging

Introduction: Over the last decade, ADAR1p150 has been linked by our research team and others to malignant progenitor reprogramming and therapeutic resistance in a broad array of malignancies. However, ADAR1p150's role in accelerated hematopoietic stem and progenitor cell (HSPC) aging in response to inflammatory cytokines had not been clearly elucidated. Here, we investigate ADAR1 splice isoform switching and the ADAR1p150 inhibitory effects of Rebecsinib on human HSPC function in normal humanized aged normal bone marrow (aNBM) HSPC mouse models. Methods: Whole transcriptome RNA sequencing (RNA-seq) was utilized with splice isoform and editome analysis to detect ADAR1 splice isoform switching and adenosine to inosine RNA editing. Rebecsinib (17S-FD-895), a pre-IND drug candidate, has shown promising potential as an inhibitor of ADAR1p150-mediated RNA editing ( Crews, Ma...Jamieson. Cell Stem Cell 2023), which is an important therapeutic target in patients with myelofibrosis or acute myeloid leukemia that overexpress ADAR1p150 compared with ADAR1p110. Human CD34 + cells, isolated from aged normal bone marrow (aNBM) from patients undergoing elective hip replacement surgery, were transduced with a lentiviral ADAR1-nano-luciferase reporter and transplanted into NSG-SGM3 adult mice that secrete human SCF, IL-3, and GM-CSF cytokines that support normal hematopoiesis. After engraftment confirmation peripheral blood human CD45 flow cytometric assessment and IVIS 200 system imaging of ADAR1-nano-luc-GFP reporter expressing HSPC engraftment, the mice were randomly divided into vehicle-treated and Rebecsinib-treated groups (15mg/kg, twice a week for two weeks). Peripheral blood, bone marrow, and spleen were collected upon treatment completion for analysis. Results: By HSPC RNA-seq, we detected an imbalance between ADAR1p150 and ADAR1p110 expression together with increased RNA editing and splicing alterations using whole transcriptome RNA sequencing (RNA-seq) of aged compared with young bone marrow hematopoietic stem and progenitor cells (HSPCs). However, the functional relevance of ADAR1 splice isoform switching had not been evaluated in human-aged normal bone marrow HSPC xenograft (PDX) mouse models nor had this cytokine-induced HSPC aging been reversed. Flow cytometric analysis and immunohistochemistry staining revealed that Rebecsinib treatment at a dose of 10mg/kg, twice a week for two weeks, effectively spared normal human immune cells (CD3+, CD14+, and CD19+ cells). Remarkably, Rebecsinib treatment not only preserved Hematopoietic Stem (CD34+CD38-) cells and Progenitor (CD34+CD38+) cells in peripheral blood, bone marrow, and spleen but also promoted their expansion. These findings demonstrate the potential of Rebecsinib to enhance the retention of HSPC populations in the bone marrow niche. Conclusions: Our pre-clinical experiments utilizing RNA-seq, a novel ADAR1-nano-luciferase-GFP reporter, and aNBM PDX mouse models provide compelling evidence supporting the potential of Rebecsinib to restore the balance of ADAR1p150:p110 ratios and engraftment in humanized aNBM HSPC mouse models. These findings highlight Rebecsinib as a promising therapeutic candidate for targeting leukemia and myelofibrosis while preserving essential HSPC populations.