scholarly journals Whole-Genome CRISPR Screen Reveals the Mechanism of Relapse in Patient-Derived Cells Representing High-Risk Paediatric ALL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3952-3952
Author(s):  
Katarzyna Szoltysek ◽  
Helen Blair ◽  
Sirintra Nakjang ◽  
Ricky Tirtakusuma ◽  
Mankaran Singh ◽  
...  
Keyword(s):  
High Risk ◽  
Research Funding ◽  
Ex Vivo ◽  
Mtor Inhibitors ◽  
Whole Genome ◽  
Nsg Mice ◽  
Genome Wide ◽  
Crispr Screen ◽  
Dexamethasone Resistance

Acute lymphoblastic leukemia (ALL) is the most common type of childhood leukaemia. Recently improved risk stratification resulted in therapy optimization and extended survival for the majority of cases. Unfortunately, there is still a significant number of patients either relapsing or not responding to treatment with response to glucocorticoids being one of the most important prognostic indicators of treatment outcome. In order to investigate the mechanism of dexamethasone resistance, we performed genome-wide CRISPR screens in patient derived xenotransplant (PDX) material from t(17;19)-positive ALL. Primary material was obtained from the patient at the presentation and at relapse stage of disease and corresponding PDX samples were generated in immunocompromised NSG mice. PDX cells were lentivirally transduced with the CRISPR knockout pooled 'Brunello' library and then subjected to dexamethasone pressure both ex vivo and in vivo. For the in vivo screen, CRISPR-modified cells were intrafemorally injected into immunodeficient NSG mice followed by either 7.5mg/kg dexamethasone or vehicle treatment. In parallel, PDXs were co-cultured with mesenchymal and endothelial-like human stromal cells generated from human bone marrow-derived iPSCs. Data analysis performed with the MAGeCKFlute software identified the glucocorticoid receptor gene NR3C1 as a main driver of chemoresistance-mediated relapse in this high-risk ALL. Notably, a homozygous deletion of NR3C1 was present in the relapse PDX sample. Furthermore, we identified that loss of the NR3C1 gene in those cells was associated with an inferior engraftment potential in the absence of dexamethasone. Interestingly, the whole-genome CRISPR screen in the relapse sample identified BCL2 and several genes associated with the mTOR pathway as crucial for leukaemic propagation. Knockout of NR3C1 in the diagnostic PDX also established dexamethasone resistance and further enhanced the already significant sensitivity towards mTOR inhibitors. To explore a potential synergism between BCL2 and mTOR inhibition, we assessed the effect of the BCL2 inhibitor ABT-199 and several mTOR inhibitors in both presentation and relapse PDX samples. PDX samples were co-cultured with MSCs and treated with drug combinations in a matrix format for 96 hrs followed by high-throughput fluorescence microscopy-based analysis. These experiments revealed substantial synergism of ABT-199 and mTOR inhibitors associated with increased cell death and prolonged growth inhibition in both presentation and relapse samples. In conclusion, our studies (i) demonstrate that genome-wide CRISPR screens are feasible in PDX material both ex vivo and in vivo, (ii) provide an explanation for the relative rarity of NR3C1 mutations in relapsed material and (iii) identify drug combinations effective in both diagnostic and relapse PDX for further preclinical evaluation. Disclosures Vormoor: Abbvie (uncompensated): Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding; AstraZeneca: Research Funding.

scholarly journals CD46 Is Amplified in High-Risk Myeloma with Gain of Chromosome 1q and Selectively Targeted By a Novel Anti-CD46 Antibody-Drug Conjugate

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 384-384
Author(s):  
Daniel W. Sherbenou ◽  
Blake T. Aftab ◽  
Yang Su ◽  
Christopher R. Behrens ◽  
Arun P. Wiita ◽  
...  
Keyword(s):  
Single Dose ◽  
High Risk ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Ex Vivo ◽  
Chromosome 1Q ◽  
Nsg Mice ◽  
Antibody Drug

Abstract Background: Multiple myeloma (MM) is incurable by standard approaches, with relapse and development of treatment resistance inevitable in all patients. We previously identified a panel of novel macropinocytosing human monoclonal antibodies against CD46 by phage antibody library display and optimized a lead antibody for targeted drug delivery. Antibody-drug conjugates (ADCs) have recently seen proof-of-concept clinical success in Hodgkin lymphoma and breast cancer, but none is yet FDA-approved for MM. The CD46 gene is located on the long arm of chromosome 1 (1q32.2), 50Mbp from a FISH probe clinically used to identify high-risk MM and which may provide a surrogate biomarker for CD46 as a therapeutic target. Methods:We covalently conjugated the monomethyl auristatin F (MMAF) toxin to our anti-CD46 antibody via a lysosomal protease sensitive valine-citrulline linker (hereafter referred to as CD46-ADC). High Performance Liquid Chromatography analysis with hydrophobic interaction chromatography of the final conjugate showed an average drug per antibody of 3.3. CD46-ADC was evaluated for cytotoxicity in vitro in MM cell lines, in vivo with cell line xenografts in NSG mice, and ex vivo in MM patient bone marrow (BM)aspirate samples. To assess in vivo toxicity, CD46-ADC treatment was administered to transgenic mice that express the human CD46 gene under its native promoter. Results: CD46 was highly expressed on the cell surface of all 18 MM cell lines tested, and was upregulated on MM1.S cells co-cultured with the BM stromal cell line HS5. In BM aspirate samples, CD46 was highly expressed on MM cells in 100% (n=25) patients evaluated. By quantitative flow cytometry in 10 patients, the CD46 cell surface antigen density was significantly higher in patient MM cells with 1q21 gain (1q+) than those with normal 1q21 copy number (p=0.032) (Fig 1A). In patients with amp1q21 the mean CD46 antigen density on MM cells was 313,190 (SEM 68,849), compared to patients with normal 1q21 where it was 121,316 (SEM 28,352) (Fig 1A). In contrast, CD46 antigen density on normal donor (n=3) BM hematopoietic cell populations was low (antigen density range 8,443 - 23,772). Of note, higher CD46 antigen density was present on monocytes (mean 58,320, SEM 6,874) and granulocytes (mean 54,439, SEM 10,688) relative to the other populations (Fig 1B). CD46-ADC potently inhibited proliferation in all 14 MM cell lines tested (EC50 range of 150 pM - 5 nM) (Fig 1C). On BM stromal cells, CD46-ADC had EC50 >100 nM for patient-derived BM61 (generated via culture of CD138-negative BM) cells and no effect on HS5 cells in concentrations tested up to 150 nM. CD46-ADC eliminated MM growth in two orthometastatic xenograft models. In one model, MM1.S cell line xenografts expressing firefly luciferase grown in NSG mice were treated once every 3-4 days at either 4 mg/kg or 0.8 mg/kg for 4 injections, or with a single dose of 4 mg/kg (Fig 2A). Control groups were treated with vehicle, nonbinding ADC or naked antibody (CD46-mAb). CD46-ADC 4 mg/kg (4 dose) eliminated bioluminescent activity throughout the duration of the study (Fig 2B), and all mice survived to study discontinuation (Fig 2C). The single dose and low dose groups showed elimination of bioluminescence, but all mice relapsed (Fig 2B-C). In patient BM aspirate samples, CD46-ADC induces apoptosis and cell death in primary MM cells ex vivo (EC50 <10 nM), but did not affect the viability of non-tumor mononuclear cells (MNCs). For in vivo toxicity study, human CD46 transgenic mice were treated with a single IV bolus injection of 6 mg/kg CD46-ADC and showed no body weight loss or overt side effects for 14 days. At study discontinuation (day 14), histologic analysis of major organs showed no notable tissue damage. Conclusion: We have identified a novel functional antigen, CD46, for ADC targeting of MM, with unique potential for high-risk and relapsed/refractory disease that has genomic amplification at the CD46 gene locus and are in dire need of therapy. The novel CD46-ADC is highly potent and selective in eliminating MM cells (cell lines and primary tumor cells) in preclinical models. CD46 genomic gain on chromosome 1q correlates with antigen amplification, andindentifies a potential biomarker based on a clinical FISH test that can be used for patient stratification. Thus, our study could lead directly to the application of a novel ADC therapeutic for treating MM. Disclosures Aftab: Onyx Pharmaceuticals, Inc.: Research Funding; Atara Biotherapeutics, Inc.: Employment, Equity Ownership; Omniox, Inc.: Research Funding; CytomX: Research Funding; Cleave Biosciences, Inc.: Research Funding. Wiita:Onyx Pharmaceuticals: Research Funding; Omniox, LLC: Research Funding; Cleave Biosciences: Research Funding; Quadriga Biosciences: Research Funding. Wolf:Celgene: Honoraria; Telomere Diagnostics: Consultancy; Takeda: Honoraria; Amgen: Honoraria; Pharmacyclics: Honoraria. Martin:Sanofi: Research Funding; Amgen: Research Funding.

A Whole Genome In Vivo Crispr Screen in Primary ALL Predicts Leukaemic Relapse

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2619-2619
Author(s):  
Katherine Dormon ◽  
Elda S Latif ◽  
Matthew Bashton ◽  
Deepali Pal ◽  
Matthew Selby ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Conflicts Of Interest ◽  
Gene Set Enrichment Analysis ◽  
Whole Genome ◽  
Competitive Situation ◽  
Nsg Mice ◽  
A Genome ◽  
Crispr Screen

Abstract Although paediatric acute lymphoblastic leukaemia (ALL) has a favourable prognosis, a number of cases will invariably relapse. One of the major problems associated with relapse is drug resistance, in particular to glucocorticoids, the mainstay of ALL treatment. Examining the underlying mechanisms is complicated by clonal heterogeneity within a patient and the potential impact of the leukaemic niche. To address mechanisms of drug resistance in a patient-relevant setting, we performed a genome-wide in vivo CRISPR screen in primary ALL material. To that end, we took advantage of primografted material from patient L707, who initially presented with a Dexamethasone (DEX) sensitive t(17;19) ALL, but relapsed 5 months after initial diagnosis. We transduced DEX sensitive presentation cells with the full genome GeCKOv2 CRISPR library, before transplantation into immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Mice were subsequently treated with DEX by oral gavage (15mg/kg for 5 weeks, 10mg/kg thereafter). DNA from several engrafted sites in the mouse was extracted and PCR amplified before being sequenced on the Illumina HiSeq2500. Changes in pool complexity were analysed using MaGEcK software to determine which sgRNAs were significantly enriched or depleted. By far the most significantly enriched sgRNAs were those targeting NR3C1, the gene encoding the glucocorticoid receptor. In addition, two of the top five significantly depleted sgRNAs targeted the Plexins, PLXNA1 and PLXND1. Whilst PLXNA1 is expressed at low levels, PLXND1 is highly expressed and has been linked to dexamethasone resistance. Notably, the matched relapsed material from L707 was highly DEX resistant both in tissue culture and when transplanted into NSG mice. SNP 6.0 analysis revealed a 5q deletion in the relapse, spanning 5 genes including NR3C1. Whole genome sequencing showed this was comprised of 2 deletions both targeting NR3C1, with different breakpoints for each allele. The differential gene expression between the L707 presentation and relapse established that NR3C1 was the most significant of all the genes lost at relapse, based on gene set enrichment analysis (GSEA). This contrasts with many ALL cases, where one of the downstream effectors of apoptosis is lost as opposed to NR3C1. Growth of the relapse material in vivo and in vitro was slower than the presentation in a competitive situation, but with DEX treatment the relapse phenotype began to emerge with a small percentage of cells showing a heterozygous deletion of NR3C1. These combined data strongly suggest that the NR3C1 deletion is the main driver of DEX resistance in the L707 relapse. Moreover, it proves that our in vivo CRISPR screen predicted the leukaemic relapse. These results confirm NR3C1 deletion as a driver in glucocorticoid resistance and demonstrate the power of in vivo CRISPR screens to predict mechanisms of gain of drug resistance and subsequent relapse. The parallels that can be drawn between the relapse and the CRISPR screen are striking, giving the indication that the progression from presentation to relapse may follow the same path in a patient derived xenograft setting as it did in the patient. Disclosures No relevant conflicts of interest to declare.

scholarly journals Eomes and T-Bet Expression Are Required By Mature Primary Human NK Cells for Anti-Leukemia Responses In Vivo

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 194-194
Author(s):  
Pamela Wong ◽  
Carly C. Neal ◽  
Lily Chang ◽  
Julia A Wagner ◽  
Melissa M. Berrien-Elliott ◽  
...  
Keyword(s):  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
T Box ◽  
Nsg Mice ◽  
Healthy Donors ◽  
And Function

Abstract Natural Killer (NK) cells are innate lymphoid cells that respond to hematologic cancers via cytotoxicity (perforin/granzyme and death receptors) and cytokine/chemokine production, yet the molecular determinants underlying their proliferation, function, and persistence are poorly understood. There are promising reports of pre-clinical and clinical NK cell responses to leukemia and lymphoma, which represent a nascent cellular therapy for these blood cancers. The T-box transcription factors (TFs) Eomes and T-bet are expressed by NK cells throughout their lifespan, and are required for development as evidenced by NK cell loss in Eomes and T-bet deficient mice. However, the roles of these TFs in mature human NK cell molecular programs and functions remain unclear. We hypothesized Eomes and T-bet, which are the only T-box TFs expressed in NK cells, are critical regulators of NK cell homeostasis and functionality, and are necessary for proper mature NK cell responses. To address this, we utilized the CRISPR-Cas9 system to genetically delete both Eomes and T-bet in primary human NK cells isolated from healthy donors, and investigated their role beyond guiding NK cell development, specifically in the anti-leukemia response. Gene-editing of primary human NK cells has been technically challenging, thus most reports that modified NK cells were performed with cell lines, in vitro-differentiated, or highly expanded NK cells that likely do not reflect primary human NK cell biology. Here, we introduced Cas9 mRNA and sgRNA targeting T-bet and Eomes by electroporation into unexpanded primary human NK cells isolated from healthy donors using the MaxCyte GT system. We observed highly efficient reductions of Eomes and T-bet protein expression, quantified by flow cytometry (p &lt; 0.0001, Fig A-B) without viability differences between control (sgRNA targeting TRAC, an unexpressed locus in NK cells), and Eomes/T-bet double CRISPR-edited (DKO) cells after one week in vitro. To study Eomes and T-bet in NK cell anti-leukemia response, control or DKO primary human NK cells were engrafted into NSG mice, supported with human IL-15, and challenged with K562 leukemia cells. Utilizing bioluminescent imaging to visualize leukemia burden, we observed that NK cells lacking both TFs were unable to suppress leukemia growth in vivo. To understand the mechanism responsible for impaired leukemia control, we investigated in vivo persistence and proliferation, cytotoxic effector molecule expression, as well as ex vivo degranulation and cytokine production of DKO NK cells compared to control NK cells. DKO or control human NK cells were transferred into NSG mice and supported with human IL-15. After 2-3 weeks, significantly fewer (&lt;30%) DKO NK cells persisted compared to control NK cells: spleen (5-fold decrease, control 240e3±65e3 vs DKO 47e3±15e3 NK cells, p&lt;0.01, Figure C), blood (6-fold decrease, p&lt;0.01), and liver (4-fold decrease, p&lt;0.05). Using intracellular flow cytometry, double T-bet/Eomes CRISPR-edited NK cells that lacked both Eomes and T-bet protein after in vivo transfer were identified. A proliferative defect was evident in flow-gated DKO (62±6% undivided), compared to unedited (WT) NK cells (4±2% undivided) assessed by CellTrace Violet dilution (Figure D). In addition, there were marked reductions in granzyme B and perforin protein (p&lt;0.001) in flow-gated DKO NK cells compared to controls. To assess DKO NK cell functional capacity, we performed an ex vivo functional assay on NK cells from spleens of the NSG mice as effectors, and K562 targets or IL-12/15/18 stimulation for 6 hours. Degranulation to K562 targets was impaired (p&lt;0.05), and IFN-γ production was reduced (p&lt;0.0001) after cytokine stimulation in flow-gated DKO NK cells (Figure E). Thus, CRISPR-editing of unexpanded, primary human NK cells revealed that Eomes and T-bet are required by mature human NK cells for their function and homeostasis, distinct from their role in development. This is translationally relevant, as defects in proliferation and function of human DKO NK cells manifested markedly reduced response against human leukemia cells in vivo in xenografts. These findings expand our understanding of key molecular regulators of mature NK cell homeostasis and function, with the potential to provide new avenues to enhance NK cell therapy. Figure 1 Figure 1. Disclosures Berrien-Elliott: Wugen: Consultancy, Patents & Royalties: 017001-PRO1, Research Funding. Foltz-Stringfellow: Kiadis: Patents & Royalties: TGFbeta expanded NK cells; EMD Millipore: Other: canine antibody licensing fees. Fehniger: HCW Biologics: Research Funding; Compass Therapeutics: Research Funding; Affimed: Research Funding; ImmunityBio: Research Funding; Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; Kiadis: Other; OrcaBio: Other; Indapta: Other.

scholarly journals Pharmacological Induction of NOXA Sensitizes High-Risk B Cell Acute Lymphoblastic Leukemia Cells to Venetoclax

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-18
Author(s):  
Klaudyna Fidyt ◽  
Julia Cyran ◽  
Agata Pastorczak ◽  
Nicholas T. Crump ◽  
Angelika Muchowicz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Cell Lines ◽  
Culture System ◽  
Ex Vivo ◽  
Chromatin Accessibility ◽  
Leukemic Cells ◽  
Nsg Mice ◽  
Thioredoxin System

Background: Venetoclax (VEN), a specific BCL2 inhibitor, exerts anti-leukemic effects in various high-risk (HR) B-ALL subtypes, such as ALL with mixed lineage leukemia (MLL) gene rearrangements (MLLr ALL) (PMID: 26711339), Philadelphia chromosome-positive (Ph+) (PMID 30546081) or hypodiploid B-ALL (PMID 30862722). Nevertheless, despite high rationale for targeting BCL2 in these subtypes of B-ALL, VEN monotherapy is not effective enough to completely eliminate leukemic cells. For this reason identification of other drugs that could sensitize leukemic cells to VEN may become beneficial treatment strategy in HR ALL. Previously, we showed that the enzymes of the thioredoxin system are upregulated in primary B-ALL cells and that auranofin (AUR), a thioredoxin reductase inhibitor, effectively kills leukemic cells in vitro and in vivo. Importantly, elements of the thioredoxin system are not only balancing redox homeostasis within the cells, but may also interact with other pathways, including anti-apoptotic signaling. Considering above, we hypothesized that AUR may potentiate VEN efficacy in HR B-ALL. Methods: To evaluate cytostatic/cytotoxic effects of VEN+AUR combination by MTT assay and propidium iodide (PI)-staining we used HR B-ALL cell lines, including SEM (MLLr ALL), BV-173 (Ph+ ALL) and NALM-16 (hypodiploid ALL). Patient derived xenograft cells (PDX) were generated through long-term propagation of primary B-ALL samples in immune-deficient NSG mice. Ex vivo drug testing in co-culture system was performed using primary bone marrow-derived mesenchymal stem cells (BM-MSC) and murine stromal OP9 cell line. NOXA genomic knockout (KO) in SEM cells was established by CRISPR/Cas9 system. Chromatin accessibility within PMAIP1 gene (encodes for NOXA) was detected using ATAC-seq. Results: We observed that AUR sensitizes HR B-ALL cell lines to VEN, as determined by MTT and PI-staining. Further, we mimicked the bone marrow support of stromal cells towards B-ALL and evaluated its impact on the response to VEN+AUR. For this reason we employed an ex vivo co-culture system of B-ALL PDX cells with primary BM-MSC or an OP9 cell line. In all tested PDX samples representing diagnostic/relapsed MLLr ALL (n=8), Ph+ ALL (n=2) and Ph-like ALL (n=2) we observed synergistic effect of this combination (Fig. 1A). Next, we determined the efficacy of VEN+AUR combination in vivo using a PDX model of MLLr B-ALL. We observed that administration of VEN+AUR diminished the progression of leukemia during a 3 week-long treatment more effectively than any single drug alone, which reflected in longer survival of NSG mice (Fig. 1B). Subsequently, we aimed to uncover the mechanism responsible for the synergistic action of VEN+AUR. In cells treated with both drugs we observed enhanced caspase activation and changes in the levels of BCL2 family proteins involved in apoptotic signaling. In particular, we found that AUR strongly upregulates a pro-apoptotic NOXA protein, both in HR B-ALL cell lines and in MLLr ALL PDX samples (Fig. 1C). To evaluate whether NOXA induction is functionally relevant for the cell death mediated by VEN+AUR, we generated SEM cells with a NOXA genomic KO. Lack of NOXA significantly abolished VEN-single agent as well as VEN+AUR combination cytotoxicity, demonstrating its dependence on NOXA expression (Fig. 1D). We then showed that NOXA is regulated at the transcriptional level, as co-treatment with AUR and the transcription inhibitor, actinomycin D, abolished AUR-mediated NOXA induction at mRNA and protein levels in SEM cells. Additionally, to test whether AUR-treatment itself provokes changes in chromatin accessibility within the NOXA encoding gene (PMAIP1) we performed ATAC-seq. We observed a clear increase in accessibility at PMAIP1 in response to AUR, which correlated with transcriptional induction of NOXA. Moreover, ChIP-qPCR revealed that increased ATAC peaks within PMAIP1 were associated with an increase in H3 lysine 27 acetylation (H3K27ac) - an epigenetic mark associated with open chromatin conformation. Conclusions: Our results demonstrate that FDA-approved drug, AUR, is a promising candidate to be used in combination with VEN for the therapy of HR B-ALL subtypes. Importantly, NOXA induction by AUR plays a central role in the VEN+AUR synergistic cytotoxicity. More studies elucidating the mechanism of NOXA upregulation by AUR are underway. Disclosures Milne: OxStem Oncology (OSO), a subsidiary company of OxStem Ltd.: Other: Founding shareholder .

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

Blood ◽  
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 &lt;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 &gt;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&lt;.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&lt;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.

scholarly journals BMT CTN 1803: Haploidentical Natural Killer Cells (CSTD002) to Prevent Post-Transplant Relapse in AML and MDS (NK-REALM)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1955-1955
Author(s):  
Sumithira Vasu ◽  
Nelli Bejanyan ◽  
Steven Devine ◽  
Elizabeth Krakow ◽  
Elizabeth Krakow ◽  
...  
Keyword(s):  
High Risk ◽  
Nk Cells ◽  
Cumulative Incidence ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
Marrow Transplant ◽  
Free Survival ◽  
Blood And Marrow Transplant ◽  
Relapse Rates

Background and Rationale: Relapse remains the leading cause of treatment failure for patients with high-risk acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) undergoing allogeneic blood or marrow transplantation (BMT). Although relapse rates vary based on patient population, age, and conditioning intensity, relapse is experienced in at least 30-50% after conventional BMT in high-risk AML/MDS. Initial safety and post-BMT relapse risk reduction results are reported by investigators at MD Anderson Cancer Center in a phase I study of ex vivo-expanded, donor-derived, haploidentical natural killer (NK)-cell infusion in conjunction with haploBMT. Of 13 patients with high-risk myeloid malignancies treated with NK cells, no infusion reactions or dose-limiting toxicities occurred and only 1 patient, treated at the lowest dose of 1×105 cells/kg, relapsed (Ciurea, Blood 2017). This experience supports investigation of CSTD002, a product derived from haploidentical donor NK cells and expanded ex vivo using plasma membrane (PM21) nanoparticles bearing membrane-bound IL-21 and 4-1BBL. This study represents a public-private partnership between the sponsor (Kiadis Pharma) and the Blood and Marrow Transplant Clinical Trials Network (BMT CTN), leveraging existing National Institutes of Health-supported clinical trials infrastructure to conduct a complex cellular immunotherapy trial. We used contemporary, unpublished data from the Center for International Blood and Marrow Transplant Research registry to determine baseline relapse rates that informed the statistical design. Doses of NK cells expanded by a novel method and exceeding those previously achieved in most published studies will be given in the peri-transplant period to test the hypothesis that haploidentical NK cells can mediate an effective anti-leukemia response. Trial Design and Methods: BMT CTN 1803 is a phase II, single-arm, open-label, multicenter trial designed to investigate the safety and efficacy of CSTD002 for the treatment of patients with high-risk AML or MDS undergoing haploBMT. An initial safety run-in phase will precede enrollment into the full study of approximately 60 patients. Major inclusion criteria of patients and donors are listed in the Table. Peripheral blood will be drawn from the donor to start the NK-cell expansion approximately 5 weeks before the planned haploBMT. Patients will receive intravenous (IV) melphalan 140 mg/m2 (100 mg/m2 for patients ≥60 years old) on Day -7; fludarabine 40 mg/m2 IV on Days -7, -6, -5, and -4; and 2 Gy of total body irradiation on Day -3. Donor bone marrow will be harvested and given on Day 0. Three doses of CSTD002 will be administered IV on Days -2, +7, and +28, relative to the haploBMT. The recommended dose of CSTD002 for administration will be formulated at 1×108 NK cells/kg of recipient body weight. Graft-versus-host disease (GVHD) prophylaxis is post-transplantation cyclophosphamide with tacrolimus and mycophenolate mofetil. The primary endpoint is cumulative incidence of relapse at 1 year post haploBMT in patients receiving at least 1 infusion of CSTD002. Secondary endpoints are safety and tolerability of CSTD002; overall survival; non-relapse mortality; relapse-free survival; GVHD-free survival; cumulative incidence of acute GVHD and chronic GVHD; hematologic recovery; donor-cell engraftment; primary and secondary graft failure; overall incidence of toxicity; and cumulative incidence of infections including cytomegalovirus re-activation and symptomatic BK virus hemorrhagic cystitis. Exploratory endpoints are systemic immunosuppression-free survival; immune reconstitution at Days 28, 100, and 365 post haploBMT; proportion of patients with detectable minimal residual disease at Days 28 and 100 post haploBMT; feasibility of administering the planned CSTD002 doses; and impact of NK-cell alloreactivity on relapse and survival. Disclosures Vasu: Boehringer Ingelheim: Other: Travel support; Seattle Genetics: Other: Clinical trial support. Bejanyan:Kiadis Pharma: Other: advisory board. Devine:Kiadis Pharma: Other: Protocol development (via institution); Magenta Therapeutics: Other: Travel support for advisory board; My employer (National Marrow Donor Program) has equity interest in Magenta; Bristol Myers: Other: Grant for monitoring support & travel support. Krakow:Bellicum Pharmaceuticals: Research Funding; Highpass Bio: Research Funding; Magnolia Innovations: Other: Personal fees. Logan:Eisai: Other: Personal fees; Astellas: Other: Grant; Kiadis (formerly Cytosen): Other: Grant; Novartis: Other: Personal fees; Kite: Other: Grant. Luznik:Merck: Research Funding, Speakers Bureau; Genentech: Research Funding; AbbVie: Consultancy; WindMiL Therapeutics: Patents & Royalties: Patent holder. Barrett:Kiadis Pharma (formerly Cytosen): Other: Personal fees; Biologics Consulting Company: Other: Personal fees. Shan:Kiadis Pharma (formerly Cytosen): Employment. Champlin:Actinium: Consultancy; Johnson and Johnson: Consultancy; Sanofi-Genzyme: Research Funding.

scholarly journals Genome-wide CRISPR screen identifies regulators of MAPK and MTOR pathways mediating sorafenib resistance in acute myeloid leukemia

Haematologica ◽  
2020 ◽  
Author(s):  
Alisa Damnernsawad ◽  
Daniel Bottomly ◽  
Stephen E. Kurtz ◽  
Christopher A. Eide ◽  
Shannon K. McWeeney ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
Mek Inhibitors ◽  
Genome Wide ◽  
A Genome ◽  
Flt3 Mutations ◽  
Flt3 Inhibitors ◽  
Crispr Screen ◽  
Acute Myeloid

Drug resistance impedes the long-term effect of targeted therapies in acute myeloid leukemia (AML), necessitating the identification of mechanisms underlying resistance. Approximately 25% of AML patients carry FLT3 mutations and develop post-treatment insensitivity to FLT3 inhibitors, including sorafenib. Using a genome-wide CRISPR screen, we identified LZTR1, NF1, TSC1 or TSC2, negative regulators of the MAPK and MTOR pathways, as mediators of sorafenib resistance. Analyses of ex vivo drug sensitivity assays in FLT3-ITD AML patient samples revealed lower expression of LZTR1, NF1, and TSC2 correlated with sorafenib sensitivity. Importantly, MAPK and/or MTOR complex1 (MTORC1) activity were upregulated in AML cells made resistant to several FLT3 inhibitors, including crenolanib, quizartinib, or sorafenib. These cells were sensitive to MEK inhibitors, and the combination of FLT3 and MEK inhibitors showed enhanced efficacy, suggesting its effectiveness in AML patients with FLT3 mutations and those with resistance to FLT3 inhibitors.

RASGRP1/APTX Ratio Strongly Correlates with Clinical Response and Survival in AML Patients Treated with Tipifarnib-Bortezomib Combination.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1028-1028
Author(s):  
Stefania Paolini ◽  
Emanuela Ottaviani ◽  
Sarah Parisi ◽  
Federica Salmi ◽  
Barbara Lama ◽  
...  
Keyword(s):  
Overall Survival ◽  
Adverse Event ◽  
High Risk ◽  
Clinical Response ◽  
Stable Disease ◽  
Response Rate ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Secondary Aml

Abstract Abstract 1028 Poster Board I-50 Background: Outcome of elderly acute myeloid leukemia (AML) patients is dismal. Targeted-therapies might improve current results by overcoming drug-resistance and reducing toxicity. In particular, the farnesyl-transferase inhibitor Tipifarnib (Zarnestra®), and the proteasome inhibitor Bortezomib (Velcade®), appeared synergistic in AML cells ex vivo, and their association was shown to be safe in vivo in a phase I trial by our group. Aim We conduced a phase II study aiming to assess efficacy and toxicity of Tipifarnib-Bortezomib association in AML patients >18 years, unfit for conventional therapy, or >60 years, in relapse. Furthermore, we aimed to identify biological features potentially predictive of clinical response. In particular, we focused on the RASGRP1/APTX ratio, which was previously found to be effective in predicting treatment response in patients treated with Tipifarnib alone. Methods: Bortezomib (1.0 mg/m2) was administered as weekly infusion for three consecutive weeks (days 1, 8, 15). Tipifarnib was administered at dose of 300-600 mg BID for 21 consecutive days. Response was assessed at the end of each cycle (28 days). Patients' withdrawn was planned in case of progression or stable disease after six cycles. Real-time quantitative-PCR (q-PCR) was used for RASGRP1/APTX quantification. Results: Eighty patients were enrolled (47 male). Median age was 71 years (43-89) and WBC at diagnosis was 4.2 × 109/L (0.5- 42.1). Thirty-two out of 80 patients had a secondary-AML, 14 had a high risk cytogenetic and 42 were previously untreated. Seventy-five patients actually initiated the treatment, 62 completed at least the first cycle while 13 early dropped out for non-leukemia related adverse event. Nine patients achieved complete remission (CR), 1 patients obtained a partial response (PR) and in 2 cases an hematological improvement (HI) was documented for an overall response rate (ORR) of 19%. Eighteen had progressive disease (PD) and the remaining showed stable disease (SD). Median time to response was 112 days, corresponding to 4 cycles (range 2-14). Marrow response (CR+PR) was significantly associated with overall survival (OS) (p<0.0001). RASGRP1/APTX was evaluated before treatment initiation on bone marrow (BM) and/or peripheral blood (PB). The median RASGRP/APTX value on BM was 15.3 (15-19.8) in responder patients and 2.2 (0.5-25.9) in non responders, respectively (p=0.00006). Its median value on PB was 31.6 (19.3-35.5) in responders and 6.4 (0.5-27.1) in non responders, respectively (p=0.00001). Interestingly, no marrow responses were recorded in patients with marrow RASGRP1/APTX ratio <8, while the response rate was 43% in patients with RASGRP1/APTX >8 (p<0.0001). Finally, RASGRP1/APTX levels significantly correlated with OS (p=0.001) with a median OS of 490 days and 162 days in patients with RASGRP1/APTX >8 and <8 respectively. Conversely, there was no correlation between cytogenetics, secondary AML, previous treatment and response or overall survival. Toxicity was overall mild, the most common adverse event being febrile neutropenia. Permanent treatment interruption due to Tipifarnib-Bortezomib related adverse events occurred in 13/75 (17%) of patients. With a median follow-up of 122 days (range 9-737), 57/75 (76%) patients are dead and 18/75 (24%) are alive, six of which in CR. Conclusion: We conclude that the clinical efficacy of the combination Tipifarnib-Bortezomib was similar to what reported for Tipifarnib alone. However, noteworthy, we could confirm that the RASGPR1/APTX BM or PB level is an effective predictor of response. Though higher RASGRP1/APTX is relatively rare (∼10% of cases), Tipifarnib (±Bortezomib) may represent an important option in a subset of high risk/frail AML patients. Acknowledgments: Supported by BolognAIL, AIRC, European LeukemiaNET, COFIN, FIRB 2006, Fondazione del Monte di Bologna e Ravenna. Disclosures: No relevant conflicts of interest to declare.

MLN9708 Elicits Pharmacodynamic Response in the Bone Marrow Compartment and Has Strong Antitumor Activity in a Preclinical Intraosseous Model of Plasma Cell Malignancy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1834-1834 ◽  
Author(s):  
Edmund Lee ◽  
Bret Bannerman ◽  
Michael Fitzgerald ◽  
Jennifer Terkelsen ◽  
Daniel Bradley ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Antitumor Activity ◽  
Plasma Cell ◽  
Research Funding ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Tumor Burden ◽  
Human Multiple Myeloma ◽  
Cell Malignancy

Abstract Abstract 1834 Poster Board I-860 Introduction The clinical success of VELCADE® (bortezomib) for Injection has validated the proteasome as a therapeutic target for the treatment of human cancer. The novel proteasome inhibitor MLN9708 is a potent, reversible, and specific inhibitor of the b5 site of the 20S proteasome identified in preclinical studies. MLN9708 is currently in human clinical development for both hematological and non-hematological malignancies. Here we describe the pharmacodynamic (PD) response of MLN9708 in the murine bone marrow compartment and its strong antitumor activity in an intraosseous xenograft model of plasma cell malignancy. Materials MLN9708 immediately hydrolyzes to MLN2238, the biologically active form, upon exposure to aqueous solutions or plasma. MLN2238 was used for all preclinical studies described below. Methods It has been previously shown that double transgenic iMycCa/Bcl-XL mice develop de novo plasma cell malignancies (J. Clin. Invest. 113:1763-1773, 2004) in which neoplastic plasma cell development is driven by the targeted expression of the transgene Myc (c-myc; myelocytomatosis oncogene) and Bcl-x (Bcl2l1; encodes the oncoprotein Bcl-XL). DP54 is a plasma cell tumor cell line derived from the bone marrow of a syngeneic mouse previously inoculated with an iMycCa/Bcl-XL tumor (Cancer Res. 67:4069-4078, 2007). In vitro, DP54 cells express both the Myc and Bcl-XL transgenes, various plasma cell and B-cell markers including CD38, CD138 and B220, and has gene expression profile very similar to human multiple myeloma. To establish a preclinical intraosseous model of plasma cell malignancy for efficacy studies, freshly dissociated DP54-Luc cells (constitutively expressing firefly luciferase under a mouse Ig-k promoter) were aseptically injected into the bone marrow space of the upper shaft of the right tibia of NOD-SCID mice. Once tumor growth has been established, mice were randomized into treatment groups and then treated intravenously (IV) with vehicle, bortezomib (at 0.8 mg/kg twice weekly [BIW]) or MLN2238 (at 11 mg/kg BIW) for 3 consecutive weeks. Tumor burden was measured by bioluminescent imaging. Results MLN2238 strongly inhibited proteasome activity in the blood and bone marrow compartments of mice (maximum b5 inhibition of 84% and 83%, respectively). In vivo, when DP54 cells were aseptically injected into the bone marrow space of the mouse tibia, signs of bone erosion in the tibia, femur and cranial sagittal sultures (as determined by ex-vivo mCT imaging) were observed which resembled osteolytic lesions frequently seen in human multiple myeloma. Dissemination of DP54-Luc cells after intratibia inoculations were detected by in vivo bioluminescent and confirmed by ex vivo imaging where luminescent tumor nodules were detected in the spleen, kidneys, intestine, lymph nodes and bones including right tibia, spine and cranium. To assess the antitumor activity of MLN2238 in the bone marrow compartment, an efficacy study was performed using the DP54-Luc intraosseous xenograft model of plasma cell malignancy. Tumor burden (bioluminescence), osteolytic lesions (mCT) and overall survival after treatment with bortezomib and MLN2238 will be presented. Conclusion The novel proteasome inhibitor MLN9708 demonstrates strong activity in the bone marrow compartment in vivo. MLN9708 is currently in human clinical development for both hematological and solid tumor indications. Disclosures Lee: Milllennium: Employment, Equity Ownership. Bannerman:Milllennium: Employment. Terkelsen:Milllennium: Employment. Bradley:Milllennium: Employment, Equity Ownership, Research Funding. Li:Milllennium: Employment. Li:Milllennium: Employment. Janz:Milllennium: Research Funding. Van Ness:Milllennium: Research Funding. Manfredi:Milllennium: Employment. Kupperman:Milllennium: Employment.

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