Single Cell Mass Cytometry of Dysregulated Signaling Networks in Myeloproliferative Neoplasms and Secondary Acute Myeloid Leukemia

Abstract Abstract 703 The classic myeloproliferative neoplasms (MPNs) polycythemia vera (PV), essential thrombocythemia, and primary myelofibrosis (PMF) are frequently associated with the JAK2 V617F mutation or other genetic alterations in members of the JAK-STAT axis. These mutations have been shown to cause hyperactivated JAK-STAT signaling in cell lines and mouse models. How accurately these models recapitulate human MPN pathogenesis remains uncertain, as in vivo signaling in MPNs is likely modulated by other genetic changes and regulatory dynamics. In addition, the phenotypic changes that accompany transformation of chronic MPNs to secondary acute myeloid leukemia (sAML) have not been well characterized. While targeted inhibitors of JAK2 have shown activity in MPNs, the incomplete responses observed clinically have called into question the utility of JAK2 as a therapeutic target, suggesting that dysregulation of other signaling pathways may be important in MPN pathogenesis. Therefore, a more complete assessment of JAK-STAT and related signaling pathways in MPNs is needed. Mass cytometry is a novel technology that merges aspects of flow cytometry with mass spectrometry – cells are labeled with antibodies conjugated to elemental isotope reporters and then analyzed on the CyTOF mass cytometer. Each mass channel is distinct, such that no compensation is required, thus circumventing the spectral limitations of fluorescence-based flow cytometry and enabling the simultaneous measurement of 30+ parameters at the single cell level. We have utilized this approach to examine multiple signaling effectors in cell populations throughout hematopoietic differentiation. Our initial experiment included samples from three MPN patients (PV, PMF, post-PV sAML), and one normal donor. Cells were exposed to nine different perturbation conditions ex vivo, including cytokines and the JAK1/2 inhibitor ruxolitinib. Cells were stained with a panel of 17 surface markers and 13 dynamic intracellular signaling effectors and analyzed on the CyTOF. Single cell data was uploaded into SPADE (spanning-tree analysis of density-normalized events), which distills multidimensional data down to interconnected cell subsets and creates 2D tree plots based on shared surface marker expression. These plots identified recognizable cell subsets, including hematopoietic stem/progenitors (HSPCs) and myeloid and lymphoid lineage subsets. Heat maps were constructed to depict the relative induction of each intracellular marker in response to each condition. In the HSPC compartment, several expected responses were observed, particularly in PV. Erythropoietin-mediated activation of STAT3 and thrombopoietin (Tpo)-mediated activation of STAT3/5 were enhanced in PV committed progenitors. On a broader level, PV HSPCs exhibited heightened signaling sensitivities involving several cytokines and downstream effectors. Notably, CREB and S6 phosphorylation were strongly induced by Tpo, G-CSF, and IL-3. Ruxolitinib pre-treatment markedly inhibited signaling mediated by Tpo in PV CD34+ cells, indicating that the HSPC compartment can be effectively targeted by ex vivo JAK1/2 inhibition. In contrast to PV, PMF HSPCs exhibited lesser sensitivity to cytokine stimulation. In several instances, such as IL-3 induction of pSTAT5, the responses were in fact suppressed compared to normal. CD34+ HSPC from the sAML patient generally exhibited subnormal signaling responses. However, widespread hyperactivation following exposure to the phosphatase inhibitor pervanadate (PVO4) was observed in sAML CD34+ cells, suggesting that these signaling pathways were activated in vivo, but that feedback inhibition in response to persistent activity led to downregulation of their ex vivo inducibility. Based on these preliminary findings, we hypothesize that the fundamental chronic phase MPN state is one of heightened cytokine signaling sensitivity, while the advanced phase (especially sAML) state is one of tonic or constitutive downstream signaling activity with persistent feedback inhibition on cytokine signaling pathways. To test this hypothesis, experiments with a larger cohort of MPN samples are currently underway. These studies will provide a comprehensive framework of altered signaling in MPNs and provide deeper insights into the role of targeted therapy for MPNs. Disclosures: No relevant conflicts of interest to declare.

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Mass Cytometry Analysis Of Myelofibrosis and Secondary Acute Myeloid Leukemia Reveals Constitutive and Cytokine Induced Signaling Abnormalities With Differential Sensitivities To Ruxolitinib

Blood ◽

10.1182/blood.v122.21.1610.1610 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1610-1610

Author(s):

Daniel A.C. Fisher ◽

Gregory K. Behbehani ◽

Garry P. Nolan ◽

Sean C. Bendall ◽

Stephen T. Oh

Keyword(s):

Acute Myeloid Leukemia ◽

Single Cell ◽

Myeloid Leukemia ◽

Ex Vivo ◽

Jak2 V617f ◽

Signaling Molecules ◽

Mass Cytometry ◽

Secondary Acute Myeloid Leukemia ◽

Acute Myeloid

Abstract The classic myeloproliferative neoplasms (MPNs), including primary and secondary myelofibrosis (MF), are frequently associated with the JAK2 V617F mutation or other genetic alterations in members of the JAK-STAT axis. These mutations induce hyperactivated JAK-STAT signaling in cell lines and mouse models. Other mutations have been found recurrently in human MPNs, however, including mutations associated with transformation of chronic MPNs to secondary acute myeloid leukemia (sAML). The aggregate effects of these mutations on in vivo myeloproliferative signaling and disease phenotypes are not yet well understood. While targeted inhibitors of JAK2 have shown activity in MPNs, evidence of a selective effect on the underlying malignant clone has been lacking. These findings suggest that dysregulation of other signaling molecules may be important in MPN pathogenesis, and that therapeutic targeting of these molecules could be beneficial. Therefore, a more complete assessment of JAK-STAT and related signaling pathways in MPNs is needed. Mass cytometry is a novel technology that merges flow cytometry with mass spectrometry and enables the simultaneous measurement of 30+ parameters at the single cell level. We utilized this approach to examine bone marrow or peripheral blood samples from four MF patients and five sAML patients, as well as three normal controls. Cells were exposed to five different perturbation conditions ex vivo, including the cytokines thrombopoietin (TPO) and G-CSF, and/or the JAK1/2 inhibitor ruxolitinib. Cells were stained with a panel of 18 surface markers and 16 intracellular signaling effectors and analyzed on a CyTOF mass cytometer. Single cell data was analyzed using traditional gating strategies, as well as with SPADE (spanning-tree analysis of density-normalized events), which distills multidimensional data down to interconnected cell subsets based on shared surface marker expression. These methods grouped cells into distinct cell populations including hematopoietic stem/progenitors (HSPCs) and myeloid and lymphoid lineage subsets. Heat maps were constructed to depict basal and induced activation of each intracellular marker. MF and sAML patient samples exhibited abnormal cytokine-induced signaling that varied in a patient-specific manner. HSPC responses to TPO ranged from hyposensitive to hypersensitive compared with normal. Markedly abnormal basal (unstimulated) signaling in the HSPC compartment was observed in all MF and sAML patients. Each sample exhibited increased total IkBα and/or pCREB, typically accompanied by elevated phosphorylation of one or more additional signaling molecules. Both basal elevation and cytokine hypersensitivity were frequently observed within a MAP kinase signaling axis represented by pERK and downstream targets pCREB and pS6. Conversely, repressed basal STAT1 phosphorylation was observed in all patients. These abnormalities were not exclusive to sAML versus MF, nor specific to patients with or without the JAK2 V617F mutation. Widespread dysregulation of total IkBα, pERK-pCREB-pS6, and pSTAT1 suggest that these signaling effectors may be characteristic of a general myeloproliferative signaling phenotype. Ex vivo ruxolitinib effectively inhibited the majority of observed hypersensitive cytokine-stimulated signaling effects. In contrast, most basal signaling elevations were insensitive to ruxolitinib. The signaling molecules most frequently insensitive to ruxolitinib were those most frequently basally elevated in MF and sAML HSPC, namely pCREB and total IkBα. In contrast, phosphorylation of the JAK2 substrates STAT3 and STAT5 was almost always sensitive to ruxolitinib. These results suggest that targeting of myeloproliferative signaling pathways either downstream or independent of JAK2 activity may be valuable for improved treatment of MF and sAML patients. Ongoing experiments are focused on determining whether constitutive ruxolitinib-insensitive signaling abnormalities can be identified in patients treated with ruxolitinib in vivo, which may underlie the incomplete responses observed clinically. Taken together, these approaches will provide a deeper understanding of altered signaling networks in the context of targeted therapies in MPNs. Disclosures: Oh: Incyte: Consultancy, Research Funding, Speakers Bureau.

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Overcoming the UCB HSCs –Derived NK Cells Dysfunctionality through Harnessing RAS/MAPK, IGF-1R and TGF-β Signaling Pathways

10.21203/rs.3.rs-98506/v1 ◽

2020 ◽

Author(s):

Alireza Shokouhifar ◽

Gholamreza Anani Sarab ◽

Mahboubeh Yazdanifar ◽

Mohammad Fereidouni ◽

Masoumeh Nouri ◽

...

Keyword(s):

Nk Cells ◽

Signaling Pathways ◽

Mononuclear Cells ◽

Ex Vivo ◽

Carcinoma Cells ◽

Hematopoietic Stem ◽

Generating Functional ◽

Cd34 Cells ◽

Erythroleukemia Cell

Abstract BackgroundNatural killer (NK) cells differentiated from umbilical cord blood (UCB) hematopoietic stem cells (HSCs) may be more suitable for cell-based immunotherapy compared to NK cells from adult donors. This is due to opportunity to choose alloreactive donors and potentially more robust in vivo expansion. However, the cytotoxicity of UCB-HSC derived NK cells against cancer cells might be suboptimal. To overcome this obstacle, we attempted to generate NK cells with potent antitumor activity by targeting RAS/MAPK, IGF-1R and TGF-β signaling pathways.MethodsThe CD34+ cells isolated from human UCB mononuclear cells through MACS with purity of (≥90%) were used to be differentiated into NK cells. After 21 days of induction with SFTG36, IS721 and IL-15/Hsp70 media, NK cells phenotype was studied and their cytotoxicity against K562 human erythroleukemia cell and SKOV3 ovarian carcinoma cells was analyzed.ResultsThe induced NK cells treated with SFTG36/I721 and SFTG36/IS721 growth factor cocktail expressed a phenotype with CD56+16+CD3- and NKG2D+ with mean fluorescence intensity (MFI) of 92.7%±1.45-168.00±19.20 and 93.23%±0.75-168.66±20.00 respectively. These NK cells once activated by IL-15, demonstrated a higher cytotoxicity against K562 (≥90%) (P ≤ 0.001) and SKOV3 tumor cells (≥65%) (P ≤ 0.001) compared to IL-15/Hsp70 activated NK cells.Conclusion The differentiation of ex vivo-expanded CD34+ cells through manipulation of RAS/MAPK, IGF-1R and TGF-β signaling pathways is an efficient approach for generating functional NK cells that can be used for cancer immunotherapy.

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Overcoming the UCB HSCs –Derived NK cells Dysfunction through Harnessing RAS/MAPK, IGF-1R and TGF-β Signaling Pathways

Cancer Cell International ◽

10.1186/s12935-021-01983-z ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Alireza Shokouhifar ◽

Gholamreza Anani Sarab ◽

Mahboubeh Yazdanifar ◽

Mohammad Fereidouni ◽

Masoumeh Nouri ◽

...

Keyword(s):

Nk Cells ◽

Signaling Pathways ◽

Mononuclear Cells ◽

Ex Vivo ◽

Hematopoietic Stem ◽

Generating Functional ◽

Gm Csf ◽

Erythroleukemia Cells ◽

Cd34 Cells

Abstract Background The natural killer (NK) cells differentiated from umbilical cord blood (UCB) hematopoietic stem cells (HSCs) may be more suitable for cell-based immunotherapy compared to the NK cells from adult donors. This is due to the possibility to choose alloreactive donors and potentially more robust in vivo expansion. However, the cytotoxicity of UCB-HSC-derived NK cells against cancer cells might be suboptimal. To overcome this obstacle, we attempted to generate NK cells with potent antitumor activity by targeting RAS/MAPK, IGF-1R and TGF-β signaling pathways using IL-15, IGF-1 and SIS3 respectively. Methods The CD34 + cells were isolated from human UCB mononuclear cells through magnetic activation cell sorting (MACS) with purity of (≥ 90%) and were subjected to differentiate into NK cells. After 21 days of induction with SFTG36 (SCF, FLt-3L, TPO, GM-CSF, IL-3 and IL-6), IS721 (IGF-1, SIS3, IL-7 and IL-21) and IL-15/Hsp70 media, NK cells phenotypes were studied and their cytotoxicity against K562 human erythroleukemia cells and SKOV3 ovarian carcinoma cells was analyzed. Results The NK cells induced in SFTG36/IS721 medium were selected for activation due to their higher expression of CD56 + 16 + CD3 − (93.23% ± 0.75) and mean fluorescence intensity (MFI) of NKG2D + (168.66 ± 20.00) and also a higher fold expansion potential (11.893 ± 1.712) compared to the other groups. These cells once activated with IL-15, demonstrated a higher cytotoxicity against K562 (≥ 90%; P ≤ 0.001) and SKOV3 tumor cells (≥ 65%; P ≤ 0.001) compared to IL-15/Hsp70-activated NK cells. Conclusions The differentiation of ex vivo expanded CD34 + cells through manipulation of RAS/MAPK, IGF-1R and TGF-β signaling pathways is an efficient approach for generating functional NK cells that can be used for cancer immunotherapy.

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Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

Nature Communications ◽

10.1038/s41467-021-24730-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

David S. Fischer ◽

Meshal Ansari ◽

Karolin I. Wagner ◽

Sebastian Jarosch ◽

Yiqi Huang ◽

...

Keyword(s):

T Cells ◽

Single Cell ◽

Rna Sequencing ◽

Ex Vivo ◽

Severe Disease ◽

Human Leukocyte ◽

Cell Receptor ◽

Single Cell Rna Sequencing

AbstractThe in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

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A novel mouse model based on intersectional genetics enables unambiguous in vivo discrimination between plasmacytoid and other dendritic cells and their comparative characterization

10.1101/2022.01.07.475382 ◽

2022 ◽

Author(s):

Michael Valente ◽

Nils Collinet ◽

Thien-Phong Vu Manh ◽

Karima Naciri ◽

Gilles Bessou ◽

...

Keyword(s):

Dendritic Cells ◽

Steady State ◽

Mouse Model ◽

Single Cell ◽

Ex Vivo ◽

High Specificity ◽

Type I ◽

Specific Expression ◽

Physiological Functions

Plasmacytoid dendritic cells (pDC) were identified about 20 years ago, based on their unique ability to rapidly produce copious amounts of all subsets of type I and type III interferon (IFN-I/III) upon virus sensing, while being refractory to infection. Yet, the identity and physiological functions of pDC are still a matter of debate, in a large part due to their lack of specific expression of any single cell surface marker or gene that would allow to track them in tissues and to target them in vivo with high specificity and penetrance. Indeed, recent studies showed that previous methods that were used to identify or deplete pDC also targeted other cell types, including pDC-like cells and transitional DC (tDC) that were proposed to be responsible for all the antigen presentation ability previously attributed to steady state pDC. Hence, improving our understanding of the nature and in vivo choreography of pDC physiological functions requires the development of novel tools to unambiguously identify and track these cells, including in comparison to pDC-like cells and tDC. Here, we report successful generation of a pDC-reporter mouse model, by using an intersectional genetic strategy based on the unique co-expression of Siglech and Pacsin1 in pDC. This pDC-Tomato mouse strain allows specific ex vivo and in situ detection of pDC. Breeding them with Zbtb46GFP mice allowed side-by-side purification and transcriptional profiling by single cell RNA sequencing of bona fide pDC, pDC-like cells and tDC, in comparison to type 1 and 2 conventional DC (cDC1 and cDC2), both at steady state and during a viral infection, revealing diverging activation patterns of pDC-like cells and tDC. Finally, by breeding pDC-Tomato mice with Ifnb1EYFP mice, we determined the choreography of pDC recruitment to the micro-anatomical sites of viral replication in the spleen, with initially similar but later divergent behaviors of the pDC that engaged or not into IFN-I production. Our novel pDC-Tomato mouse model, and newly identified gene modules specific to combinations of DC types and activations states, will constitute valuable resources for a deeper understanding of the functional division of labor between DC types and its molecular regulation at homeostasis and during viral infections.

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Whole-Genome CRISPR Screening Identifies N-Glycosylation As an Essential Pathway and a Potential Novel Therapeutic Target in CALR-Mutant MPN

Blood ◽

10.1182/blood-2021-149218 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 58-58

Author(s):

Anna E. Marneth ◽

Jonas S. Jutzi ◽

Angel Guerra-Moreno ◽

Michele Ciboddo ◽

María José Jiménez Santos ◽

...

Keyword(s):

Ex Vivo ◽

Myeloproliferative Neoplasms ◽

Lectin Binding ◽

Single Gene ◽

Whole Genome ◽

Sequencing Data ◽

Therapeutic Implications ◽

Platelet Counts ◽

Secretion Pathways

Abstract Somatic mutations in the ER chaperone calreticulin (CALR) are frequent and disease-initiating in myeloproliferative neoplasms (MPN). Although the mechanism of mutant CALR-induced MPN is known to involve pathogenic binding between mutant CALR and MPL, this insight has not yet been exploited therapeutically. Consequently, a major deficiency is the lack of clonally selective therapeutic agents with curative potential. Hence, we set out to discover and validate unique genetic dependencies for mutant CALR-driven oncogenesis. We first performed a whole-genome CRISPR knockout screen in CALR Δ52 MPL-expressing hematopoietic cells to identify genes that were differentially required for the growth of cytokine-independent, transformed CALR Δ52 cells as compared to control cells. Using gene-set enrichment analyses, we identified the N-glycan biosynthesis, unfolded protein response, and the protein secretion pathways to be amongst the most significantly differentially depleted pathways (FDR q values <0.001, 0.014, and 0.025, respectively) in CALR Δ52 cells. We performed a secondary CRISPR pooled screen focused on significant pathways from the primary screen and confirmed these findings. Strikingly, seven of the top ten hits in both screens were linked to protein N-glycosylation. Four of those genes encode proteins involved in the enzymatic activity of dolichol-phosphate mannose synthase (DPM1, DPM2, DPM3, and MPDU1). This enzyme synthesizes dolichol D-mannosyl phosphate, an essential substrate for protein N-glycosylation. Importantly, these findings from an unbiased whole-genome screen align with prior mechanistic studies demonstrating that both the N-glycosylation sites on MPL and the lectin-binding sites on CALR Δ52 are required for mutant CALR-driven oncogenesis. We next performed single gene CRISPR Cas9 validation studies and found that DPM2 is required for CALR Δ52-mediated transformation, as demonstrated by increased cell death, reduced p-STAT5 and decreased MPL cell-surface levels, when Dpm2 is knocked out. Importantly, cells cultured in cytokine-rich medium were unaffected by DPM2 loss. Upon cytokine withdrawal, a sub-clone of non-edited Dpm2WT CALR Δ52 cells grew out, further demonstrating requirement for DPM2 for the survival of CALR Δ52 cells. Additionally, we observed a >50% reduction in ex vivo myeloid colony formation of murine CalrΔ52 Dpm2 ko bone marrow (BM) compared with CRISPR-Cas9 non-targeting controls, with non-significant effects on CalrWT BM cells. To enable clinical translation, we performed a pharmacological screen targeting pathways significantly depleted in our CRISPR screens. Screening 70 drugs, we found that the N-glycosylation pathway was the only pathway in which all tested compounds preferentially killed CALR Δ52 transformed cells. We then treated primary Calr Δ52/+ mice with a clinical grade N-glycosylation (N-Gi) inhibitor and found platelet counts (Sysmex) to be significantly reduced (vehicle 3x10 6/mL, N-Gi 1x10 6/mL after 18 days, p<.0001). Concordantly, the proportion of megakaryocyte erythrocyte progenitors (MEPs) was significantly reduced in CalrΔ52 BM (p=0.03). We next performed competitive BM transplantation assays using CD45.2 UBC-GFP MxCre CalrΔ52 knockin and CD45.1 mice. We found that mice treated with N-Gi had significantly reduced platelet counts (vehicle 1440x10 6/mL, N-Gi 845x10 6/mL, p=0.005) as well as significantly reduced platelet chimerism (vehicle 55%, N-Gi 27%, p<0.001), indicating a distinct vulnerability of CalrΔ52 over WT cells. Finally, we interrogated RNA-sequencing data from primary human MPN platelets. We found N-glycosylation-related pathways to be significantly upregulated in CALR-mutated platelets (n = 13) compared to healthy control platelets (n = 21), highlighting the relevance of our findings to human MPN. In summary, using unbiased genetic and focused pharmacological screens, we identified the N-glycan biosynthesis pathway as essential for mutant CALR-driven oncogenesis. Using a pre-clinical MPN model, we found that in vivo inhibition of N-glycosylation normalizes key features of MPN and preferentially targets CalrΔ52 over WT cells. These findings have therapeutic implications through inhibiting N-glycosylation alone or in combination with other agents to advance the development of clonally selective therapeutic approaches in CALR-mutant MPN. AEM and JSJ contributed equally. Figure 1 Figure 1. Disclosures Mullally: Janssen, PharmaEssentia, Constellation and Relay Therapeutics: Consultancy.

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Isoform Selective HDAC8 Inhibitor (OCH3) Shows Potency in Selectively Targeting Primary AML Cells

Blood ◽

10.1182/blood-2021-151599 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2944-2944

Author(s):

Amal Mechaal ◽

Amudha Ganapathy ◽

Dolores Mahmud ◽

Taha Y Taha ◽

Rajeev Ranjan ◽

...

Keyword(s):

Growth Inhibition ◽

Ex Vivo ◽

Vehicle Control ◽

Leukemia Cells ◽

Apoptotic Cells ◽

Histone H4 ◽

Inhibitory Effects ◽

Growth Inhibitory ◽

Cd34 Cells

Abstract The treatment outcomes for patients diagnosed with acute myeloid leukemia (AML) are still dismal. Recent advances in understanding AML indicate that the lack of efficacy is primarily due to non-specificity of currently used chemotherapeutics targeting both leukemic stem/progenitor cells (LSC) and normal hematopoietic stem cells (HSC). Thus, a critical barrier is the identification of innovative therapies that selectively target LSC. Histone deacetylase 8 (HDAC8) has been shown to enhance p53 protein deacetylation, which results in inactivation of p53, promoting LSC survival. We hypothesize that enzymatic/non-enzymatic role of HDAC8 is critical for LSC survival but not for HSCs. Then, we characterized our two tetrahydroisoquinoline (TIQ)-based selective HDAC8 inhibitors (HDAC8i) BIP and OCH3 for growth inhibition, apoptosis, activation of caspase 3, integrity of mitochondrial membrane potential (MMP), and acetylation of histone H4 in human leukemia cell lines. The growth inhibitory effects observed in cell lines were validated using bone marrow (BM) or peripheral blood (PB) cells from AML patients. Colony forming cell (CFC) assays were performed using AML BM/PB cells treated with OCH3 or BIP. OCH3 and BIP were also tested for hematotoxicity using normal CB CD34+ cells. Furthermore, we compared class I HDAC isoform engagement in human normal cord blood (CB) CD34+ cells and in SET-2 leukemia cells using our novel photoreactive probe TH1143. In CD34+ cells, TH1143 had higher level of engagement for HDAC1 and 2, whereas engagement of HDAC3 and 8 was minimal. In SET-2 cells, HDAC3 and HDAC8 displayed relatively higher engagement with TH1143 indicating HDAC engagement is likely cell type specific. The biological efficacies of OCH3 at 50uM and BIP at 25uM were noted to exert >50% growth inhibition in KG1 and in K562 leukemia cells. Both OCH3 and BIP significantly increased the number of apoptotic cells and there was an enhanced active caspase-3 activity. Furthermore, OCH3 and BIP treated cells displayed lower red/green ratio in comparison to control, indicative of poor MMP and depolarization to induce apoptosis (Table 1.a). OCH3 and BIP were further validated by using BM/PB cells from AML patients showing growth inhibition. This was also accompanied by increase in apoptotic cells by OCH3 and BIP. In contrast to BIP, OCH3 spared CB CD34+ cells as demonstrated by notably lower growth inhibition, apoptotic cells vs control when compared with primary AML cells from patients. Both OCH3 and BIP displayed minimal inhibition of CFU growth in CD34+ cells. However, HDAC8i induced significant CFU growth inhibition in primary AML samples suggesting that HDAC8i spares normal CFU progenitors but not leukemia progenitors (Table 1.b). Notably, both BIP and OCH3 lack ability to exert acetylation of histone H4, unlike broad spectrum HDAC inhibitor TSA (MFI with OCH3=0.96±0.03, BIP=0.77±0, TSA =1.63±0.15) which is consistent with isoform selectivity of OCH3 and BIP. The leukemia growth inhibitory effects at LSC level was demonstrated using ex vivo OCH3 treated AML patient derived BM/PB cells transplantation in humanized immunodeficient NSGS mice. After 10 to 12 weeks of transplantation mice receiving untreated AML cells had 7.73±2.18% while with OCH3 treatment mice had 4.84±1.37% human CD34+ leukemia cells, a 38% reduction in CD34+ leukemia cells, despite only a single ex vivo exposure to OCH3. Furthermore, in a second model, NSGS humanized mice were transplanted (IV) with primary leukemia cells from AML patients and after 4 weeks injected (IP) with OCH3 or vehicle control. After 12 weeks of transplantation in this second model human primary AML cell burden was 5.74±1.31% (OCH3) and 18.13±12.76% (vehicle control), while mice transplanted with normal CD34+ cells treated similarly with OCH3 or vehicle control displayed no detectable inhibition of human myeloid cell chimerism (OCH3:12.28 ± 3.31% vs vehicle control: 17.92±11.96%). Taken together, our data indicate that HDAC8 isoform inhibitor, OCH3 displayed significant inhibition of primary AML patient derived leukemia cells growth in vitro and in vivo in contrast to normal CD34+ cells. Selective inhibition of HDAC8 is sufficient to cause growth inhibition in primary AML progenitors including LSCs in vivo while sparing normal HSCs thus offer opportunities for further development of HDAC8i as new experimental therapeutics in AML. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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APR-246 induces early cell death by ferroptosis in acute myeloid leukemia.

Haematologica ◽

10.3324/haematol.2020.259531 ◽

2021 ◽

Author(s):

Rudy Birsen ◽

Clement Larrue ◽

Justine Decroocq ◽

Natacha Johnson ◽

Nathan Guiraud ◽

...

Keyword(s):

Lipid Peroxidation ◽

Cell Death ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Ex Vivo ◽

Lipid Peroxides ◽

Death Process ◽

Glutathione Biosynthesis ◽

Acute Myeloid

APR-246 is a promising new therapeutic agent that targets p53 mutated proteins in myelodysplastic syndromes and in acute myeloid leukemia. APR-246 reactivates the transcriptional activity of p53 mutants by facilitating their binding to DNA target sites. Recent studies in solid cancers have found that APR-246 can also induce p53-independent cell death. In this study, we demonstrate that AML cell death occurring early after APR-246 exposure is suppressed by iron chelators, lipophilic antioxidants and inhibitors of lipid peroxidation, and correlates with the accumulation of markers of lipid peroxidation, thus fulfilling the definition of ferroptosis, a recently described cell death process. The capacity of AML cells to detoxify lipid peroxides by increasing their cystine uptake to maintain major antioxidant molecule glutathione biosynthesis after exposure to APR-246 may be a key determinant of sensitivity to this compound. The association of APR-246 with induction of ferroptosis (either by pharmacological compounds, or genetic inactivation of SLC7A11 or GPX4) had a synergistic effect on the promotion of cell death, both in vivo and ex vivo.

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852 Trifunctional NKp46/CD16a-NK cell engager targeting CD123 overcomes acute myeloid leukemia resistance to ADCC

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.852 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A893-A893

Author(s):

Laurent Gauthier ◽

Angela Virone-Oddos ◽

Jochen Beninga ◽

Benjamin Rossi ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Antitumor Activity ◽

Nk Cells ◽

Myeloid Leukemia ◽

Nk Cell ◽

Ex Vivo ◽

Activating Receptors ◽

Acute Myeloid

BackgroundThere is a clear need for targeted therapies to treat acute myeloid leukemia (AML), the most common acute leukemia in adults. CD123 (IL-3 receptor alpha chain) is an attractive target for AML treatment.1 However, cytotoxic antibody targeting CD123 proved insufficiently effective in a combination setting in phase II/III clinical trials.2 T-cell engagers targeting CD123 displayed some clinical efficacy but were often associated with cytokine release syndrome and neurotoxicity.3 Interest in the use of NK cells for therapeutic interventions has increased in recent years, as a potential safer alternative to T cells. Several NK-cell activating receptors, such as CD16a, NKG2D, and the natural cytotoxicity receptors NKp30 and NKp46, can be targeted to induce antitumor immunity. We previously reported the development of trifunctional NK-cell engagers (NKCEs) targeting a tumor antigen on cancer cells and co-engaging NKp46 and CD16a on NK cells.4MethodsWe report here the design, characterization and preclinical development of a novel trifunctional NK cell engager (NKCE) targeting CD123 on AML cells and engaging the activating receptors NKp46 and CD16a on NK cells. The CD123 NKCE therapeutic molecule was engineered with humanized antibodies targeting NKp464 and CD123.5 We compared CD123-NKCE and a cytotoxic ADCC-enhanced antibody (Ab) targeting CD123, in terms of antitumor activity in vitro, ex vivo and in vivo. Pharmacokinetic, pharmacodynamic and safety profile of CD123-NKCE were evaluated in non-human primate (NHP) studies.ResultsThe expression of the high affinity Fc gamma receptor CD64 on patient-derived AML cells inhibited the ADCC of the Ab targeting CD123 in vitro and ex vivo, but not the antitumor activity of CD123-NKCE. CD123-NKCE had potent antitumor activity against primary AML blasts and AML cell lines, promoted strong NK-cell activation and induced cytokine secretion only in the presence of AML target cells. Its antitumor activity in mouse model was greater than that of the comparator antibody. Moreover, CD123-NKCE had strong and prolonged pharmacodynamic effects in NHP when used at very low doses, was well-tolerated up to high 3 mg/kg dose and triggered only minor cytokine release.ConclusionsThe data for activity, safety, pharmacokinetics, and pharmacodynamics provided here demonstrate the superiority of CD123-NKCE over comparator cytotoxic antibody, in terms of antitumor activity in vitro, ex vivo, in vivo, and its favorable safety profile, as compared to T-cell therapies. These results constitute proof-of-principle for the efficacy of CD123-NKCE for controlling AML tumors in vivo, and provide consistent support for their clinical development.ReferencesEhninger A, Kramer M, Rollig C, et al. Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia. Blood Cancer J 2014;4:e218.Montesinos P, Gail J Roboz GJ, et al. Safety and efficacy of talacotuzumab plus decitabine or decitabine alone in patients with acute myeloid leukemia not eligible for chemotherapy: results from a multicenter, randomized, phase 2/3 study. Leukemia 2021;35(1):62–74.Uy GL, Aldoss I, Foster MC, et al. Flotetuzumab as salvage immunotherapy for refractory acute myeloid leukemia. Blood 2021;137(6):751–762.Gauthier L, Morel A, Anceriz N, et al. Multifunctional natural killer cell engagers targeting NKp46 trigger protective tumor immunity. Cell 2019;177(7):1701–13.Jin L, Lee EM, Ramshaw HS, et al. Monoclonal antibody-mediated targeting of CD123, IL-3 receptor alpha chain, eliminates human acute myeloid leukemic stem cells. Cell Stem Cell 2009;5:31–42.

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