Targeting the Inflammatory Niche in MDS By Tasquinimod Restores Hematopoietic Support and Suppresses Immune-Checkpoint Expression in Vitro

Abstract Introduction Myelodysplastic syndromes (MDS) belong to the most common hematological neoplasms in the elderly population, characterized by ineffective hematopoiesis, peripheral cytopenia and the risk of transformation into acute myeloid leukemia. There is increasing evidence that an aberrant innate immune response and a proinflammatory bone marrow (BM) microenvironment play a critical role in the pathogenesis of MDS. The alarmin S100A9, a key player for regulation of inflammatory responses, has been shown to be elevated in MDS patients. It directs an inflammatory cell death (pyroptosis) by increased NF-kB mediated transcription and secretion of proinflammatory, hematopoiesis-inhibitory cytokines and production of reactive oxygen species. Tasquinimod (TASQ, Active Biotech) is a novel, oral small molecular drug with S100A9 inhibitory activity and it is currently investigated in a phase Ib/IIa trial in relapsed/refractory multiple myeloma (NCT04405167). TASQ has demonstrated anti-angiogenic, antitumor and immunomodulatory properties in a broad range of preclinical solid tumor models; however, little is known about its effects in myeloid malignancies. Aim We investigated the role of S100A9 in cellular models of MDS and the potential of TASQ to target S100A9 within the MDS stroma in vitro. Methods Immunohistochemical staining of S100A9, CD271+ mesenchymal stromal cells (MSCs), CD68+ macrophages and CD66b+ neutrophils in BM tissues from MDS patients and healthy donors was performed with multiplex immunohistochemistry and analyzed with the VECTRA imaging system. MSCs from patients with either low-risk MDS, CMML or age-adjusted healthy donors were exposed to S100A9 (1.5µg/ml) in the presence or absence of TASQ (10µM). Subsequently, TLR4 downstreaming molecules such as IRAK1, gasdermin and NF-kB-p65 were analyzed by Western blot. Moreover, the mRNA expression of further proinflammatory molecules (IL-1b, IL-18, caspase1) and PD-L1 was quantified by real-time PCR. To study the impact on the hematopoietic support, MSCs were pre-treated for one week with S100A9 ± TASQ before CD34+ hematopoietic stem and progenitor cells (HSPCs) were seeded on the stromal layer. The colony formation (CAF-C) was analyzed weekly followed by a CFU-GEMM assay in methylcellulose medium. Additionally, PD-1 mRNA expression was quantified in cocultured HSPCs. Results Immunohistochemical staining of BM tissue demonstrated S100A9 expression mainly by CD66b+ neutrophils and with less extent by CD68+ macrophages. In line with this, we could not detect relevant S100A9 mRNA expression in cultured MDS or healthy MSCs in vitro. Exposure of MDS and healthy MSCs with S100A9 induced TLR4 downstream signalling as demonstrated by increased expression of IRAK1 and NF-kB-p65. We further detected a higher expression of gasdermin, an inductor of pyroptosis, in S100A9 exposed MSCs. Addition of TASQ abolished these effects and inhibited the expression of the mentioned proteins, indicating an alleviation of inflammation. Furthermore, we detected a 2-fold increase of mRNA expression of the proinflammatory cytokines IL-1b and IL-18 as well as a 5-fold increase of their activator caspase 1 in MSCs after treatment with S100A9, which could be prevented by TASQ. Interestingly, PD-L1 as a potential downstream target was induced by S100A9 by 2.5-fold and could be suppressed by TASQ to about 50%. To evaluate the impact on the hematopoietic support of MSCs, we analysed MSC/HSPC cocultures after treatment with S100A9. We observed a decreased number of cobblestone area forming cells (CAF-C) as well as reduced numbers of colonies (CFU) in a subsequent clonogenic assay, indicating a disturbed hematopoietic support by S100A9 treated MSCs. Interestingly, both the number of CAF-C and CFU could be increased by TASQ pre-treatment. Finally, the PD-1 expression in co-cultured HSPCs was regulated in the same way as its ligand in treated MSCs, nominating this interaction as a potential target of S100A9/TASQ in the MDS BM. Conclusion In summary, we provide evidence that the pathological inflammasome activation in the myelodysplastic bone marrow can be rescued by TASQ at least in part by inhibition of the S100A9 mediated TLR4 downstream signalling including NF-kB-p65 transcription and PD-L1 expression. These effects result in an improved hematopoietic support by MSCs, suggesting a potential efficacy to improve cytopenia in low-risk MDS patients. Disclosures Balaian: Novartis: Honoraria. Törngren: Active Biotech: Current Employment. Eriksson: Active Biotech: Current Employment. Platzbecker: AbbVie: Honoraria; Takeda: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria; Janssen: Honoraria; Geron: Honoraria. Röllig: Novartis: Honoraria, Research Funding; Jazz: Honoraria; Janssen: Honoraria; Bristol-Meyer-Squibb: Honoraria, Research Funding; Amgen: Honoraria; AbbVie: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Roche: Honoraria, Research Funding.

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Sequential Treatment with Rigosertib Followed By Azacitidine Maximizes the Effects on the Interferon Signaling Pathway in Hematopoietic Cells in Myelodysplastic Syndrome (MDS)

Blood ◽

10.1182/blood-2018-99-119266 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5500-5500

Author(s):

Richa Rai ◽

Stella Melana ◽

Shyamala C. Navada ◽

Rosalie Odchimar-Reissig ◽

Erin P. Demakos ◽

...

Keyword(s):

Bone Marrow ◽

P38 Mapk ◽

Research Funding ◽

Hematopoietic Cells ◽

Fold Increase ◽

Interferon Signaling ◽

Aldh Activity ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Background: MDS is a clonal stem cell disorder characterized by abnormal maturation and differentiation of hematopoietic cells. Azacitidine (AZA), a hypomethylating agent (HMA), was approved by the FDA for patients (pts) with MDS and is the standard of care as 1st line therapy for higher- risk disease. About 50% of MDS pts respond to AZA, with a median duration of response of 14 - 24 month. For those pts responding to an HMA, most either relapse or progress with worsening bone marrow failure (BMF) and have a median survival of 4 to 6 months after treatment failure. Both primary and secondary resistance remains significant clinical problems and result in poor survival. A recent study reveals that AZA activates several immunomodulatory pathways leading to activation of the antiviral defense pathway and upregulation of interferon signaling (IFN) (Chiappinelli et al, 2017). INF has multiple effects on hematopoiesis and appears highly regulated. Interferon can stimulate hematopoietic stem cells (HSC) which may be countered through activation of p38 MAPK; inhibition of the latter may improve hematopoiesis. Short-term effects of IFN stimulate myeloid and erythroid hematopoiesis. Rigosertib (RIG) is a Ras-mimetic that blocks the activation of Ras effector proteins. It has been identified as a novel anti-cancer drug which inhibits cell cycle progression and induces apoptosis of cancer cells (Athuluri-Divakar et al, 2016). It activates apoptosis related pathways by ameliorating several signaling pathways like Akt, p38, MAPK/ JNK, STAT3, β-catenin, GSK3α/β and PLK1 that can be dysregulated in MDS pts (Xu et al, 2014). In vitro, the combination of RIG with AZA (Skidan et al, AACR 2006) showed synergistic effect in inducing cell death in a sequence dependent manner requiring RIG priming. We have reported that it acts as a histone deacetylase inhibitor with chromatin modifying activity (Chaurasia et al, ASCO 2016). In a Phase I/II study the combination of RIG and AZA (NCT 01926587) produced an ORR of 85% in pts who were HMA naïve and importantly 62% in HMA failures (Navada et al, EHA 2017). The ability to reverse the clinical resistance phenotype is a novel observation with important clinical implications and understanding the mechanisms is critical to develop ways of reversing resistance. Methods: We investigated the in vitro effects of RIG combined with AZA on two cell lines one resistant and one sensitive to AZA: BW90, an AZA resistant line and MDS-L, AZA sensitive and in specimens from 2 pts treated on a clinical trial of RIG and AZA in combination, to determine effects on hematopoietic cells and IN signaling. Results: QT-PCR studies demonstrated that individual treatment of MDS-L and BW90 with RIG or combined with AZA in sequential treatment (AZA/RIG or RIG/AZA) altered chromatin remodeler (KDM2a, SET1, JMJD3 and LRWD1) transcript levels in a cell line specific context. Exposure of cells from a bone marrow from a patient prior to treatment with RIG alone or RIG/AZA failed to induce expansion of CD34+ cells and yielded maximum aldehyde dehydrogenase (ALDH) activity, a marker of primitive hematopoietic stem and progenitor cells (HSCs) (ANOVA, p=0.006). AZA alone yielded a 3.8 fold expansion of CD34+, with marked decrease in ALDH activity that was inversely proportional to the expansion of CD34+ cells. Expansion of CD34+ cells led to ≥2 fold increase in pluripotent genes (SOX2, OCT4, NANOG and ZIC3) expression levels. QT PCR studies of the effects of AZA or RIG alone or in combination on INF yielded differential effects on INFa and INFb expression as follows compared to control reported as percent fold increase for INFa - AZA 2x; RIG 0.75x, AZA/RIG 1.5x; RIG/AZA 2.5x; INFb AZA 1.3x; RIG 0.6x, AZA/RIG 1.2x; RIG/AZA 0.7x. There was a significant increase in INFa (p=<0.0001) but not INFb, with RIG priming compared to AZA alone or AZA/RIG. Conclusion: Treatments with RIG either alone or combined with AZA results in epigenetic reprogramming of pluripotency genes, and expansion of primitive HSPC. This may serve to regulate HSPC pluripotency and expression of a maturation program. A significant increase in expression of IFNa seen after exposure to RIG/AZA may lead to enhanced hematopoietic function and may explain the differences in the alternative sequence of AZA/RIG. Further studies are underway to define more specific effects on these pathways to explain the impact on the resistance phenotype. Disclosures Navada: Onconova: Research Funding. Silverman:Mount Sinai School of Medicine: Employment; Onconova Therapeutics Inc.: Patents & Royalties, Research Funding; Celgene: Research Funding; Medimmune: Research Funding; Johnson and Johnson: Research Funding; Bayer: Research Funding.

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F4/80 Identifies a Subset of Non-Mobilizable Bone Marrow HSCs Involved in Stress-Induced Hematopoiesis

Blood ◽

10.1182/blood.v128.22.569.569 ◽

2016 ◽

Vol 128 (22) ◽

pp. 569-569

Author(s):

Philip E. Boulais ◽

Nico van Rooijen ◽

Masato Tanaka ◽

Paul S. Frenette

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Research Funding ◽

Large Fraction ◽

Fold Increase ◽

Brdu Incorporation ◽

Mixed Chimerism ◽

Hematopoietic Stem ◽

Macrophage Depletion ◽

The Impact

Abstract Hematopoietic stem cell (HSCs) are commonly isolated by using cell surface markers in order to study their hierarchy and functional properties. However, even with the most rigorous methods of isolation, subsets of HSCs likely exhibit functional heterogeneity. We have found that F4/80, an adhesion G-protein coupled receptor well known as a macrophage marker, was expressed on an HSC subpopulation (50-60% of Lin− Sca1− cKit− CD150+ CD34− CD135− cells) in the bone marrow. Interestingly, F4/80 was not expressed on HSCs that have egressed in the blood and on only a small fraction (3%) of splenic HSCs. To evaluate the function of the HSC subset expressing F4/80, we transplanted competitively 200 F4/80+ or F4/80− HSCs using CD45.1/2 congenic system. We found that F4/80+ HSCs exhibited a lower engraftment potential compared to F4/80− HSCs at 16 weeks after transplantation (19.7±4.8% and 37.1±4.5% donor contribution, respectively, P=0.025), although both HSC subsets were able to sustain mixed chimerism for myeloid, B and T cells without significant alteration in lineage bias. Since F4/80+ HSCs were not found in extramedullary tissues (blood or spleen), we tested whether they could be mobilized following G-CSF or CXCR4 antagonist (AMD3100) treatment. Interestingly, F4/80+ HSCs were still retained in the bone marrow after either G-CSF- or AMD3100-induced mobilization whereas F4/80− HSCs were mobilized efficiently. However, the frequency of bone marrow F4/80+ HSCs was reduced in G-CSF-treated animals (3.3-fold, p<0.0001) while the F4/80− HSC frequency was increased (2.2-fold, p<0.0001). F4/80 could act directly to retain HSCs in the bone marrow microenvironment or it could mark a non-mobilizable pool of stem cells. To test the later possibility, we crossed transgenic mice expressing Cre recombinase knocked in the CD169 locus, a marker of bone marrow macrophage, with ROSA26-loxP-stop-loxP-tdTomato (CD169/tomato). We found that CD169/tomato selectively labelled a large fraction of F4/80+ HSCs (31.7±8.4%) by contrast to F4/80− HSCs which were, by and large, not labelled (2.1±1.2%). We next induced HSC mobilization with G-CSF. Strikingly, we found that in a manner similar to F4/80+ HSCs, CD169/tomato+ HSCs were not mobilized in the blood while their numbers were reduced in the bone marrow after G-CSF (5-fold, p=0.014). Since macrophage depletion can induce HSC mobilization (Chow et al., JEM 2011), we tested the effect of macrophage depletion using clodronate liposomes. Interestingly, F4/80+ HSCs in wild-type mice or CD169/tomato+ HSCs were not mobilized following macrophage depletion but were depleted from the bone marrow while F4/80− HSCs underwent a 3-fold expansion. The reduced HSC numbers in marrow upon mobilization by G-CSF suggested a role in survival or proliferation. To evaluate proliferation status at steady state, we performed cell cycle analyses and BrdU incorporation assay which revealed that F4/80+ HSCs significantly more proliferative than F4/80− HSCs either by cell cycle analyses (3.7-fold increase in non-G0 phase, p=0.024) or by BrdU incorporation (2-fold increase, p=0.026). We next assessed the impact of 5-flurouracil (5FU) administration a chemotherapeutic agent that kills cycling cells and induce stress hematopoietic recovery. While both F4/80+ and F4/80− HSCs were depleted 4 days after 5FU treatment, we observed that F4/80+ HSCs expanded dramatically compared to F4/80− HSCs (10-fold and 4.5-fold over F4/80− HSCs at 8 days and 12 days post-5FU, respectively) during recovery phase. Taken together, these results identify F4/80+ HSCs as a strictly resident subset of bone marrow HSCs involved in rapid recovery after hematopoietic stress. Disclosures Frenette: PHD Biosciences: Research Funding; GSK: Research Funding; Pfizer: Consultancy.

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Grb10 Mediated Akt Activation Is Required for Induction of CML Like Myeloproliferative Disease in Mice by BCR-ABL.

Blood ◽

10.1182/blood.v110.11.1012.1012 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1012-1012

Author(s):

Corinna Albers ◽

Anna L. Illert ◽

Cornelius Miething ◽

Christian Peschel ◽

Justus Duyster

Keyword(s):

Bone Marrow ◽

Tyrosine Kinases ◽

Chromosomal Translocation ◽

Control Group ◽

Myeloproliferative Disease ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Vitro Analysis ◽

The Impact

Abstract Chronic myelogenous leukaemia (CML) results from the neoplastic transformation of hematopoietic stem cells (HSC) and is characterized by a chromosomal translocation t(9;22)(q34;q11). This aberration leads to the expression of the oncogenic tyrosine kinase BCR-ABL, which mediates signals for proliferation, transformation and anti-apoptosis via various signalling pathways. Grb10, a member of the growth factor bound proteins, is known to bind activated tyrosine kinases like BCR-ABL and might be involved in the activation of the Akt signalling pathway. Here we report the impact of Grb10 for BCR-ABL mediated transformation. We exerted a siRNA based approach in combination with a murine bone marrow transplantation model. To this end we designed a MSCV based retrovirus encoding both a Grb10 microRNA and the BCR-ABL oncogene on a single construct. This approach ensured knockdowns of more than 90% in every BCR-ABL transformed cell. Methylcellulose assays demonstrated that bone marrow coexpressing Grb10 microRNA and BCR-ABL had a 4-fold decreased colony forming ability compared to control cells. We then transduced bone marrow (BM) with retrovirus coexpressing Grb10 microRNA and p185 BCR-ABL and transplanted lethally irradiated recipient Balb/C mice. The onset and progression of leukaemia was significantly delayed in mice transplanted with Grb10 microRNA and BCR-ABL compared with the BCR-ABL transduced control microRNA group. However, we were not able to completely avoid the development of leukaemia by Grb10 knockdown. Mice transplanted with the Grb10 knockdown construct showed a delayed lymphoblastic disease, positive for B220, whereas the control group developed a rapid myeloproliferative disease, characterized by CD11b and Gr-1. In vitro analysis of BaF/3 and 32D cells showed that Grb10 knockdown in combination with BCR-ABL expression leads to a reduced phosphorylation of Akt. Taken together our data demonstrate that Grb10 is required for the development of a myeloproliferative disease by BCR-ABL in mice. Hereby, Grb10 seems to be critical for the BCR-ABL induced activation of the Akt pathway. In addition, this study describes a novel approach to express an oncogene and a microRNA using a single retroviral construct. This tool can be used to systematically screen for drugable signalling targets involved in oncogenesis.

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Hematopoietic Stem Cells Are the Disease-Initiating Cells in the Myelodysplastic Syndromes

Blood ◽

10.1182/blood.v118.21.789.789 ◽

2011 ◽

Vol 118 (21) ◽

pp. 789-789 ◽

Cited By ~ 1

Author(s):

Christopher Y. Park ◽

Wendy W Pang ◽

Elizabeth Price ◽

John A. Pluvinage ◽

Stanley L. Schrier ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Low Risk ◽

In Vitro Assays ◽

Preclinical Model ◽

Functional Evaluation ◽

Hematopoietic Stem ◽

Cytogenetic Abnormalities

Abstract Abstract 789 Myelodysplastic syndrome (MDS) is a disorder of ineffective hematopoiesis presumed to originate from self-renewing clonal hematopoietic stem cells (HSC). Previous work has shown that immunophenotypic HSC from MDS patients harbor characteristic clonal cytogenetic abnormalities such as del(5q) at high levels, strongly suggesting that the HSC is the MDS-initiating cell (Tehranchi R., et al., NEJM, 363:11;1025-37, 2010); however, these studies did not examine other cytogenetic subtypes of MDS, nor did they functionally evaluate the HSC from these patients for their ability to initiate disease. We began a molecular and functional evaluation of FACS-purified HSC (Lin-CD34+CD38−CD90+CD45RA-) from MDS patients. These studies showed that the frequency of HSC in MDS bone marrow is not expanded when compared to normal, age-matched control samples. Annexin V staining also demonstrated no difference in apoptosis levels in MDS HSC compared to normal HSC; however, MDS committed myeloid progenitors (Lin-CD34+CD38+) exhibited increased apoptosis compared with normal progenitors (18% vs 39%, respectively, p <0.05). Transciptome analysis of FACS-purified MDS HSC from 10 low-risk MDS patients compared with HSC from an equal number of normal adults showed dysregulation of 3,258 mRNAs (FDR <0.1) including increased expression of genes positively associated with cell growth and proliferation (p < 0.001) and increased expression of inflammatory response genes (p < 0.015). In addition, there was widespread downregulation of numerous ribosomal protein transcripts in non-5q MDS including RPS6 and RPS19, but not RPS14 (p < 0.05). When FACS-purified HSC from a group of low-risk MDS patients were evaluated for the presence of known FISH abnormalities, the vast majority of HSC in MDS patients with defined cytogenetic abnormalities harbored clonal abnormalities (n=5, range 84–92% of total HSC) but they were not completely replaced, suggesting that non-MDS clones co-exist with MDS clones in MDS patient bone marrows. Finally, we show that FACS-purified MDS HSC can engraft irradiated, immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) pup recipients transplanted with as few as 1000 purified HSC. Long-term engraftment (assessed >12 weeks) was achieved with 50% of MDS samples tested (4/8), and resulted predominantly in myeloid engraftment with 0.8–5% total hCD45+ chimerism in the bone marrow. For each MDS HSC engrafted mouse, engraftment of the MDS clone was verified by FISH by detecting previously characterized cytogenetic abnormalities in FACS-sorted hCD45+ cells. The frequency of FISH positive cells was similar to that seen in the primary samples, suggesting no competitive disadvantage of MDS HSC in the xenotransplantation assay. Interestingly, methylcellulose colony and clonal liquid culture assays initiated from FACS-purified MDS HSC consistently grew poorly, suggesting that in vitro assays of hematopoietic potential may not accurately reflect MDS HSC biology. Together, these studies indicate that while MDS HSC are molecularly and functionally different from normal HSC, they are capable of engrafting immunodeficient NSG pups. Moreover, these data formally demonstrate that the HSC is the disease-initiating cell in MDS. This finding has significant implications for MDS research, as it provides a potential in vivo preclinical model for testing MDS therapeutics – an experimental model previously not available to investigators. Disclosures: Schrier: Locus: Consultancy.

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Anti-CD123 Chimeric Antigen Receptor T Cells (CART-123) Provide A Novel Myeloablative Conditioning Regimen That Eradicates Human Acute Myeloid Leukemia In Preclinical Models

Blood ◽

10.1182/blood.v122.21.143.143 ◽

2013 ◽

Vol 122 (21) ◽

pp. 143-143 ◽

Cited By ~ 4

Author(s):

Saar Gill ◽

Sarah K Tasian ◽

Marco Ruella ◽

Olga Shestova ◽

Yong Li ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Research Funding ◽

Conditioning Regimen ◽

Hematopoietic Stem ◽

Gm Csf ◽

Nsg Mice ◽

Cd34 Cells ◽

Society Research

Abstract Engineering of T cells with chimeric antigen receptors (CARs) can impart novel T cell specificity for an antigen of choice, and anti-CD19 CAR T cells have been shown to effectively eradicate CD19+ malignancies. Most patients with acute myeloid leukemia (AML) are incurable with standard therapies and may benefit from a CAR-based approach, but the optimal antigen to target remains unknown. CD123, the IL3Rα chain, is expressed on the majority of primary AML specimens, but is also expressed on normal bone marrow (BM) myeloid progenitors at lower levels. We describe here in vitro and in vivostudies to evaluate the feasibility and safety of CAR-based targeting of CD123 using engineered T cells (CART123 cells) as a therapeutic approach for AML. Our CAR consisted of a ScFv derived from hybridoma clone 32716 and signaling domains from 4-1-BB (CD137) and TCR-ζ. Among 47 primary AML specimens we found high expression of CD123 (median 85%, range 6-100%). Quantitative PCR analysis of FACS-sorted CD123dim populations showed measurable IL3RA transcripts in this population, demonstrating that blasts that are apparently CD123dim/neg by flow cytometry may in fact express CD123. Furthermore, FACS-sorted CD123dimblasts cultured in methylcellulose up-regulated CD123, suggesting that anti-CD123 immunotherapy may be a relevant strategy for all AML regardless of baseline myeloblast CD123 expression. CART123 cells incubated in vitro with primary AML cells showed specific proliferation, killing, and robust production of inflammatory cytokines (IFN-α, IFN-γ, RANTES, GM-CSF, MIP-1β, and IL-2 (all p<0.05). In NOD-SCID-IL2Rγc-/- (NSG) mice engrafted with the human AML cell line MOLM14, CART123 treatment eradicated leukemia and resulted in prolonged survival in comparison to negative controls of saline or CART19-treated mice (see figure). Upon MOLM14 re-challenge of CART123-treated animals, we further demonstrated robust expansion of previously infused CART123 cells, consistent with establishment of a memory response in animals. A crucial deficiency of tumor cell line models is their inability to represent the true clonal heterogeneity of primary disease. We therefore engrafted NSG mice that are transgenic for human stem cell factor, IL3, and GM-CSF (NSGS mice) with primary AML blasts and treated them with CART123 or control T cells. Circulating myeloblasts were significantly reduced in CART123 animals, resulting in improved survival (p = 0.02, n=34 CART123 and n=18 control animals). This observation was made regardless of the initial level of CD123 expression in the primary AML sample, again confirming that apparently CD123dimAML may be successfully targeted with CART123 cells. Given the potential for hematologic toxicity of CART123 immunotherapy, we treated mice that had been reconstituted with human CD34+ cells with CART123 cells over a 28 day period. We observed near-complete eradication of human bone marrow cells. This finding confirmed our finding of a significant reduction in methylcellulose colonies derived from normal cord blood CD34+ cells after only a 4 hour in vitro incubation with CART123 cells (p = 0.01), and was explained by: (i) low level but definite expression of CD123 in hematopoietic stem and progenitor cells, and (ii) up-regulation of CD123 upon myeloid differentiation. In summary, we show for the first time that human CD123-redirected T cells eradicate both primary human AML and normal bone marrow in xenograft models. As human AML is likely preceded by clonal evolution in normal or “pre-leukemic” hematopoietic stem cells (Hong et al. Science 2008, Welch et al. Cell 2012), we postulate that the likelihood of successful eradication of AML will be enhanced by myeloablation. Hence, our observations support CART-123 as a viable therapeutic strategy for AML and as a novel cellular conditioning regimen prior to hematopoietic cell transplantation. Figure 1. Figure 1. Disclosures: Gill: Novartis: Research Funding; American Society of Hematology: Research Funding. Carroll:Leukemia and Lymphoma Society: Research Funding. Grupp:Novartis: Research Funding. June:Novartis: Research Funding; Leukemia and Lymphoma Society: Research Funding. Kalos:Novartis: Research Funding; Leukemia and Lymphoma Society: Research Funding.

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Sqstm1 Is Required to Retain Hematopoietic Stem Cell/ Progenitors As a Negative Regulator of Macrophage-Dependent Inflammatory Signaling in the Bone Marrow Osteoblastic Niche

Blood ◽

10.1182/blood.v124.21.350.350 ◽

2014 ◽

Vol 124 (21) ◽

pp. 350-350

Author(s):

Kyung-Hee Chang ◽

Amitava Sengupta ◽

Ramesh C Nayak ◽

Angeles Duran ◽

Sang Jun Lee ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Research Funding ◽

Negative Regulator ◽

Wild Type ◽

Hematopoietic Stem ◽

P Retention

Abstract In the bone marrow (BM), hematopoietic stem cells and progenitors (HSC/P) reside in specific anatomical niches. Among these niches, a functional osteoblast (Ob)-macrophage (MΦ) niche has been described where Ob and MΦ (so called "osteomacs") are in direct relationship. A connection between innate immunity surveillance and traffic of hematopoietic stem cells/progenitors (HSC/P) has been demonstrated but the regulatory signals that instruct immune regulation from MΦ and Ob on HSC/P circulation are unknown. The adaptor protein sequestosome 1 (Sqstm1), contains a Phox bemp1 (PB1) domain which regulates signal specificities through PB1-PB1 scaffolding and processes of autophagy. Using microenvironment and osteoblast-specific mice deficient in Sqstm1, we discovered that the deficiency of Sqstm1 results in macrophage contact-dependent activation of Ob IKK/NF-κB, in vitro and in vivo repression of Ccl4 (a CCR5 binding chemokine that has been shown to modulate microenvironment Cxcl12-mediated responses of HSC/P), HSC/P egress and deficient BM homing of wild-type HSC/P. Interestingly, while Ccl4 expression is practically undetectable in wild-type or Sqstm1-/- Ob, primary Ob co-cultured with wild-type BM-derived MΦ strongly upregulate Ccl4 expression, which returns to normal levels upon genetic deletion of Ob Sqstm1. We discovered that MΦ can activate an inflammatory pathway in wild-type Ob which include upregulation of activated focal adhesion kinase (p-FAK), IκB kinase (IKK), nuclear factor (NF)-κB and Ccl4 expression through direct cell-to-cell interaction. Sqstm1-/- Ob cocultured with MΦ strongly upregulated p-IKBα and NF-κB activity, downregulated Ccl4 expression and secretion and repressed osteogenesis. Forced expression of Sqstm1, but not of an oligomerization-deficient mutant, in Sqstm1-/- Ob restored normal levels of p-IKBα, NF-κB activity, Ccl4 expression and osteogenic differentiation, indicating that Sqstm1 dependent Ccl4 expression depends on localization to the autophagosome formation site. Finally, Ob Sqstm1 deficiency results in upregulation of Nbr1, a protein containing a PB1 interacting domain. Combined deficiency of Sqstm1 and Nbr1 rescues all in vivo and in vitro phenotypes of Sqstm1 deficiency related to osteogenesis and HSC/P egression in vivo. Together, this data indicated that Sqstm1 oligomerization and functional repression of its PB1 binding partner Nbr1 are required for Ob dependent Ccl4 production and HSC/P retention, resulting in a functional signaling network affecting at least three cell types. A functional ‘MΦ-Ob niche’ is required for HSC/P retention where Ob Sqstm1 is a negative regulator of MΦ dependent Ob NF-κB activation, Ob differentiation and BM HSC/P traffic to circulation. Disclosures Starczynowski: Celgene: Research Funding. Cancelas:Cerus Co: Research Funding; P2D Inc: Employment; Terumo BCT: Research Funding; Haemonetics Inc: Research Funding; MacoPharma LLC: Research Funding; Therapure Inc.: Consultancy, Research Funding; Biomedical Excellence for Safer Transfusion: Research Funding; New Health Sciences Inc: Consultancy.

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Integrative Analysis of Primary SF3B1 mt Ring Sideroblasts Provides Fundamental Insights into MDS-RS Pathogenesis and Dyserythropoiesis

Blood ◽

10.1182/blood-2021-148321 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 146-146

Author(s):

Pedro Luis Moura ◽

Teresa Mortera-Blanco ◽

Isabel Juliana F Hofman ◽

Gabriele Todisco ◽

Warren W Kretzschmar ◽

...

Keyword(s):

Bone Marrow ◽

Erythroid Differentiation ◽

Integrative Analysis ◽

Model Systems ◽

Stress Factors ◽

Blue Staining ◽

Hematopoietic Stem ◽

Ring Sideroblasts ◽

The Impact

Abstract Myelodysplastic syndromes (MDS) constitute a heterogeneous group of clonal hematopoietic stem cell (HSC) disorders characterized by aberrant HSC differentiation, cytopenia, and an increased risk of progression to leukemia. The low-risk subtype MDS with ring sideroblasts (MDS-RS) is specifically characterized by expanded and ineffective erythropoiesis, with more than 80% of patients displaying mutations in the core spliceosome component SF3B1 (SF3B1 mt). A hallmark of the MDS-RS bone marrow (BM) is the progressive accumulation of ring sideroblasts (RS), erythroblasts displaying perinuclear mitochondria loaded with aberrant ferritin-iron complexes. Whilst several in vitro and in vivo model systems exist for studying the impact of SF3B1 mt on erythropoiesis and RS development, primary SF3B1 mt erythroid biology remains largely unexplored due to the inability to purify live SF3B1 mt cells or fully replicate BM conditions in vitro. To address this issue, we developed an innovative two-step method to isolate live ring sideroblasts from SF3B1 mt MDS-RS BM aspiration material with extremely high purity (as determined through droplet digital PCR-based genotyping [Fig. 1A] and morphology-based detection through Prussian blue staining [Fig. 1B,C]). Unexpectedly, evaluation of matching peripheral blood samples showed that circulating ring sideroblasts are strikingly common in MDS-RS (Fig. 1D), with their abundance being significantly positively associated with clinically-determined BM RS frequencies and serum erythropoietin levels, as well as negatively associated with hemoglobin levels. Through high-throughput Chromium 3'-based single-cell RNA sequencing (scRNAseq) analysis of purified RS, we then showed that these cells comprise a heterogeneous population encompassing all stages of the erythroid differentiation continuum, from early progenitors to orthochromatic erythroblasts (Fig. 1E). The RS transcriptome was shown to be dynamically regulated towards the maintenance of cell survival during late terminal erythroid differentiation (exemplified through parkin 1 [PINK1] expression), with SF3B1 K700E erythroblasts employing multiple strategies to preserve homeostasis despite undergoing extreme oxidative stress. These observations were confirmed through a parallel whole-transcript RNAseq investigation comprising CD34 + and GPA +-enriched samples obtained from normal bone marrow (NBM) donors and SF3B1 K700E MDS-RS patients, as well as purified RS samples. This bulk RNAseq experiment validated the RS transcriptomic signature observed in scRNAseq (Fig. 1F) and allowed for a detailed investigation of RNA splicing. SF3B1 K700E-associated alternative splicing in CD34 + and RS was consistent with previous literature, but also highly context-dependent and with substantial changes in scope and magnitude throughout erythroid differentiation (Fig. 1G-I). Finally, we substantiated these RNAseq results through Tandem Mass Tag-based semi-quantitative proteomic analysis of purified RS and GPA-enriched cells from NBM donors and MDS-RS patients. We confirmed that ring sideroblast survival is heavily dependent on redox balance modulation and suppression of ER stress via an increased dependence on glutamine, mirroring the molecular mechanisms observed in malignancy. Additionally, our data strongly indicate that the RS population is a major modulator of the MDS-RS BM microenvironment due to expression of stress factors (with particular emphasis on GDF15, erythroferrone and IL-18). In conclusion, our integrative analysis of primary RS constitutes a unique platform for the study of MDS-RS, with special interest for the investigation of potential drivers of disease severity or treatment avenues. Figure 1 Figure 1. Disclosures Kretzschmar: Vanadis Diagnostics, a PerkinElmer company.: Current Employment.

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Bone Marrow Hematopoietic Dysfunction in Untreated Chronic Lymphocytic Leukemia Is Partially Mediated By Exposure to Constituents of the Leukemic Microenvironment

Blood ◽

10.1182/blood-2018-99-118916 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3132-3132

Author(s):

Bryce Manso ◽

Kimberly Gwin ◽

Charla R Secreto ◽

Henan Zhang ◽

Wei Ding ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Board Of Directors ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Healthy Controls ◽

Advisory Committees ◽

The Impact

Abstract Peripheral immune dysfunction in B-Chronic Lymphocytic Leukemia (CLL) is well-studied and likely relates to the incidence of serious recurrent infections and second malignancies that plague CLL patients. However, the current paradigms of known immune abnormalities are not able to consistently explain these complications and it is not easy to correct CLL patient immune status. Here, we expand on our preliminary reports that demonstrate bone marrow (BM) hematopoietic dysfunction in early and late stage untreated CLL patients. We found reduced short-term functional capacity of hematopoietic progenitors in BM using colony forming unit assays (Figure 1A-C) and flow cytometry revealed significant reductions in frequencies of hematopoietic stem and progenitor cell (HSPC) populations (exemplified by Lin-CD34+ HSPCs, Figure 1D). We further report that protein levels of the transcriptional regulators HIF-1α, GATA-1, PU.1, and GATA-2 are overexpressed in distinct HSPC subsets from CLL patient BM, providing molecular insight into the basis of HSPC dysfunction. Interestingly, sustained myelopoiesis, evaluated by limiting dilution analysis in long-term culture-initiating cell (LTC-IC) assays maintained for five weeks, revealed no difference between healthy controls and CLL patients. These new data indicate that when HSPCs are removed from the leukemic microenvironment for ample in vitro culture time, they recover the ability to sustain myelopoiesis. To further assess the impact of the CLL microenvironment on HSPC biology, isolated HSPCs (CD34+ BM cells) from healthy controls were exposed in vitro to known leukemic microenvironment constituents. Exposure to TNFα, a cytokine constitutively produced by CLL B cells, resulted in rapid increases in PU.1 and GATA-2 proteins (Figure 2A-D). Similarly, addition of TNFα to the LTC-IC assay resulted in a striking ablation of myelopoiesis, even at the highest input cell concentration. Further, overexpression of PU.1 and GATA-2 were observed in HSPCs following co-culture with CLL B cells, a result that was not recapitulated when cells were exposed to IL-10, another cytokine constitutively produced by CLL B cells. These findings indicate specific components of the leukemic microenvironment are involved in HSPC modulation. Together, these findings expand on our previous observations of BM hematopoietic dysfunction in untreated CLL patients and offer new molecular insights into the contribution of the leukemic microenvironment on immunodeficiency in CLL. Disclosures Ding: Merck: Research Funding. Parikh:Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; Janssen: Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Honoraria; AstraZeneca: Honoraria, Research Funding. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Pharmacological Inhibition of Nemo-like Kinase Rescues mTOR-Mediated Translation and Primes Progenitors for Leucine-Stimulated Erythroid Expansion in Pre-Clinical Models of Diamond Blackfan Anemia

Blood ◽

10.1182/blood-2019-129570 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 455-455

Author(s):

Mark C Wilkes ◽

Jacqueline D Mercado ◽

Mallika Saxena ◽

Jun Chen ◽

Kavitha Siva ◽

...

Keyword(s):

Bone Marrow ◽

Fold Increase ◽

Healthy Controls ◽

In Vitro Activity ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Diamond Blackfan Anemia ◽

Mtor Activity

Diamond Blackfan Anemia (DBA) is associated with anemia, congenital abnormalities, and cancer. Current therapies for DBA have undesirable side effects, including iron overload from repeated red cell transfusions or infections from immunosuppressive drugs and hematopoietic stem cell transplantation. Human hematopoietic stem and progenitor cells (HSPCs) from cord blood were transduced with lentiviral shRNA against a number of ribosomal genes associated with DBA, reducing the specific ribosomal protein expression by approximately 50%. During differentiation, these cells demonstrated a DBA-like phenotype with significantly reduced differentiation of erythroid progenitors (over 80%), yet only modest (15-30%) reduction of other hematopoietic lineages. NLK was immunopurifed from differentiating HSPCs and activity was assessed by the extent of in vitro phosphorylation of 3 known NLK substrates NLK, c-Myb and Raptor. As NLK activation requires phosphorylation at Thr298, we also showed that in vitro activity correlated with intracellular NLK phosphorylation by Western blot analysis. Nemo-like Kinase (NLK) was hyperactivated in the erythroid progenitors (but not other lineages), irrespective of the type of ribosomal gene insufficiency. We extended these studies using other sources of HSPCs (fetal liver, whole blood and bone marrow), along with RPS19- and RPL11-insufficient mouse models of the disease, as well as DBA patient samples. NLK was hyperactivated in erythroid progenitors from mice (5.3- and 7.2-fold increase in Raptor phosphorylation in RPS19- and RPL-11 insufficiency respectively) and from humans (7.3- and 9.0-fold in RPS19- and RPL11-insufficiency respectively) as well as HSPCs from three DBA patient (4.8-, 4.1- and 4.2-fold increase above controls). In RPS19-insufficient human HSPCs, genetic silencing of NLK increased erythroid expansion by 2.2-fold (p=0.0065), indicating that aberrant NLK activation contributes to disease pathogenesis. Furthermore, a high-throughput inhibitor screen identified a compound that inhibits NLK (IC50:440nM) and increases erythroid expansion in murine (5.4-fold) and human (6.3-fold) models of DBA without effects on normal erythropoiesis (EC50: 0.7 µM). Identical results were observed in bone marrow CD34+ progenitors from three DBA patients with a 2.3 (p=0.0009), 1.9 (p=0.0007) and 2.1-fold (p=0.0001) increase in CD235+ erythroid progenitor population following NLK inhibition. In erythroid progenitors, RPS19-insufficiency increased phosphorylation of the mTORC1 component Raptor, reducing mTOR in vitro activity by 82%. This was restored close to basal levels (93.8% of healthy control) upon inhibition of NLK. To compensate for a reduction in ribosomes, stimulating mTOR activity with leucine has been proposed to increase translational efficiency in DBA patients. In early clinical trials, not all DBA patients have responded to leucine therapy. We hypothesize that one of the reasons might be due to NLK phosphorylation of Raptor. While leucine treatment increased mTOR activity in both RPS19-insufficient and control cells (164% of healthy controls: p=0.007 and 24% to 42% of healthy controls: p=0.0064), combining leucine with NLK inhibition increased mTOR activity in RPS19-insufficiency from 24% to 142% of control (p=0.0012). This translated to improvements in erythroid expansion of RPS19-insufficient HSPCs from 8.4% to 16.3% with leucine treatment alone, 28.4% with NLK inhibition alone, but 68.6% when leucine and NLK inhibition were combined. This 8.2-fold improvement in erythroid progenitor production indicates that identification of aberrantly activated enzymes, such as NLK, offer therapeutic promise used alone, or in combination with existing therapies, as druggable targets in the clinical management of DBA. Disclosures Glader: Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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From Bone Marrow to Mobilized Peripheral Blood Stem Cells: The Circuitous Path to Clinical Gene Therapy for Fanconi Anemia

Blood ◽

10.1182/blood-2018-99-120278 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2208-2208

Author(s):

Pamela S Becker ◽

Jennifer Adair ◽

Grace Choi ◽

Anne Lee ◽

Ann Woolfrey ◽

...

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Bone Marrow ◽

Fanconi Anemia ◽

Research Funding ◽

Hematopoietic Stem ◽

Post Infusion ◽

Clinical Gene Therapy

Abstract For decades, it has remained challenging to achieve long-term engraftment and correction of blood counts using gene-modified hematopoietic stem cells for Fanconi anemia. Toward this goal, our group conducted preclinical studies using a safety modified lentiviral vector encoding full-length cDNA for FANCA in normal and affected patient hematopoietic progenitor cells, and in a mutant mouse model that supported the IND for a gene therapy clinical trial for Fanconi anemia, complementation group A (NCT01331018). These studies led us to incorporate methods such as addition of N-acetylcysteine and hypoxic incubation during transduction. Because of the low stem cell numbers of Fanconi patients and initial difficulty with using plerixafor off-label for mobilization, we began our study with bone marrow as the source of stem cells. Due to concerns regarding secondary cancers, no conditioning was administered prior to infusion of gene-modified cells. The US Food and Drug Administration approved adult patients initially, but later permitted pediatric patient enrollment with a minimum age of 4 years. The primary objective of our phase I trial was safety. Secondary objectives included in vitro correction of mitomycin C (MMC) sensitivity, procurement of sufficient cell numbers, and ultimately, long-term correction of blood counts in recipients. Eligibility included absolute neutrophil count ≥0.5, hemoglobin ≥8, platelet count ≥20,000, lack of matched family donor, adequate organ function, and not meeting criteria for diagnosis of MDS. Our three enrolled patients were ages 22, 10, and 5 years. All demonstrated defects in the FANCA gene, with two patients sequenced and one patient diagnosed by complementation. Due to in-process learning and the later addition of plerixafor mobilization to the protocol, three different laboratory procedures were used to prepare the gene-modified product for each patient. Cell products were CD34+ selected bone marrow, bone marrow mononuclear cells depleted of red cells by hetastarch, and G-CSF and plerixafor mobilized cells depleted of red blood cells and cells bearing lineage markers, respectively. Transduction efficiencies were 17.7, 42.7 and 26.3% of colony forming cells (CFC) in 0 nM MMC, and 80, 100, and 100% of CFC in 10 nM MMC. Growth of hematopoietic colonies in MMC indicated functional correction of the FANCA defect. The 1st patient received 6.1×10e4, the 2nd 2.9×10e5, and the 3rd 4.3×10e6 CD34+ cells/kg. Serious adverse events included cytopenias in all patients, and hospital admission for fever due to viral upper respiratory infection in one patient. The patients remain alive at 46, 38, and 12 months after receipt of gene-modified cells. Due to worsening cytopenias, the third patient underwent hematopoietic cell transplant from an unrelated donor 10 months after infusion of gene-modified cells. To date, he has done well with transplant, and no indication that prior gene therapy impacted the outcome. The blood counts for the first 2 patients who have not undergone allogeneic transplant remain stable at 1,111 and 1,077 days post infusion compared to the first blood counts when they arrived at our center. For the 1st patient, vector was detectable in white blood cells (WBC) up to 21 days, in the 2nd up to 582 days, and the 3rd up to 81 days post infusion. Thus, in these patients, despite dramatic improvement in cell dose during the study, there was lack of persistence in detection of gene-modified WBCs beyond 1.5 years. A number of factors may have contributed, including lack of conditioning, in vitro cell manipulation including cytokine exposure, inability to transduce primitive hematopoietic stem cells, and paucity of long-term repopulating cells at the ages of the patients, suggesting earlier collection may be beneficial. This study is now closed to enrollment. Valuable information gained as a result of this study will contribute to future clinical gene therapy trials. Current work focuses on how to evaluate stem cell fitness prior to attempting gene therapy, minimizing manipulation required for gene correction and/or in vivo genetic correction and non-chemotherapy-based conditioning to facilitate engraftment. We would like to personally thank each patient and their families for participating in this study, as we could not have learned these lessons without their support. Disclosures Becker: GlycoMimetics: Research Funding; Abbvie: Research Funding; Amgen: Research Funding; BMS: Research Funding; CVS Caremark: Consultancy; Trovagene: Research Funding; Rocket Pharmaceuticals: Research Funding; Novartis: Research Funding; Pfizer: Consultancy; JW Pharmaceuticals: Research Funding. Adair:Miltenyi Biotec: Honoraria; RX Partners: Honoraria; Rocket Pharmaceuticals: Patents & Royalties: PCT/US2017/037967 and PCT/US2018/029983. Kiem:Rocket Pharmaceuticals: Consultancy; Homology Medicine: Consultancy; Magenta: Consultancy.

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