Flanks Of Mice Research Articles

MiR-23b-3p Inhibits the Oncogenicity of Colon Adenocarcinoma by Directly Targeting NFE2L3

Background and Aims MicroR-23b-3p (miR-23b-3p) has been found to be abnormally expressed in a variety of malignant tumors and to play a role in tumor inhibition or promotion. However, the regulatory mechanism of miR-23b-3p in COAD remains unclear. The purpose of this study was to investigate the clinical significance of miR-23b-3p expression in COAD cells and to explore its role and regulatory mechanism in the growth of COAD. Materials and Methods Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure miR-23b-3p expression in COAD tissues and cell lines. After transfecting miR-23b-3p mimics into two human COAD cell lines (SW620 and LoVo), the cell counting kit-8 (CCK-8), colony formation, and 5-ethynyl-2′-deoxyuridine (EdU) assays were used to detect cell proliferation, the Transwell assay was used to measure cell migration and invasion capacity, and flow cytometry was used to evaluate cell apoptosis in vitro. In addition, a luciferase reporter assay was used to determine whether miR-23b-3p targets NFE2L3. The downstream regulatory mechanisms of miR-23b-3p action in COAD cells were also investigated. For in vivo tumorigenesis assay, COAD cells stably overexpressing miR-23b-3p were injected subcutaneously into the flank of nude mice to obtain tumors. Results Significantly decreased expression of miR-23b-3p was detected in COAD tissues and cell lines. Exogenous miR-23b-3p expression inhibited cell proliferation, migration, and invasion and promoted cell apoptosis of COAD cells in vitro. Nuclear factor erythroid 2 like 3 (NFE2L3) was identified as a direct target gene of miR-23b-3p. In addition, reintroduction of NFE2L3 partially abolished the anticancer effects of miR-23b-3p on COAD cells. Furthermore, miR-23b-3p overexpression hindered the growth of COAD cells in vivo. Conclusion miR-23b-3p inhibited the oncogenicity of COAD cells in vitro and in vivo by directly targeting NFE2L3, suggesting the importance of the miR-23b-3p/NFE2L3 pathway in the development of COAD.

Abstract P201: Zenocutuzumab is an effective HER2/HER3 Biclonics® antibody in cancers with NRG1 fusions

Abstract Neuregulin 1 (NRG1) fusion proteins are oncogenic drivers in multiple cancer types, including non-small cell lung cancer, pancreas adenocarcinoma, and other solid tumors. NRG1 fusion proteins bind to HER3 and signal through HER2/HER3 heterodimers, leading to oncogenic transformation. Zenocutuzumab is an HER2/HER3 humanized IgG1 bispecific antibody that specifically and potently blocks NRG1 fusion-driven signaling of the heterodimeric complex. Zenocutuzumab binds via its anti-HER2 monovalent Fab arm to domain I of HER2. Docking of zenocutuzumab in this position increases the effective affinity of the anti-HER3 Fab arm, which binds to domain 3, a critical epitope that blocks binding of the HER3 ligand NRG1. Consequently, zenocutuzumab displaces NRG1 binding to HER3, even at high ligand concentrations, keeping HER3 in an inactive state and blocking HER2/HER3 heterodimerization and NRG1-mediated signaling. The clinical efficacy and safety of zenocutuzumab were recently demonstrated in patients with NRG1 fusion-driven cancers who were enrolled in the ongoing global multicenter eNRGy study and a global early access program NCT02912949 (J Clin Oncol 39;15_suppl; abstract 3003). Treatment with zenocutuzumab led to rapid and major radiologic tumor regression and biomarker responses in heavily pretreated patients with multiple NRG1 fusion-positive cancers with several different N-terminal fusion partners. Zenocutuzumab has been enhanced for antibody-dependent cell-mediated cytotoxicity (ADCC) to recruit and bolster immune effector cells to aid in tumor elimination. Here we explored the different Fc-mediated immune effector functions of zenocutuzumab. In several cancer cell lines, zenocutuzumab induced Fc-mediated dose-dependent ADCC and antibody-dependent cellular phagocytosis. Insertion of ATP1B1-NRG1 and SLC3A2-NRG1 into immortalized pancreatic ductal epithelial cells (H6c7) led to activation of the HER3 oncogenic signaling pathways including AKT. The H6c7-SLC3A2-NRG1 cells formed tumors when implanted in the subcutaneous flank of immune compromised mice (after about 10 weeks). Furthermore, a dependency of the transformed cell on this oncogene for growth and survival was also observed. Zenocutuzumab effectively inhibited proliferation of these NRG1 fusion-expressing, transformed cells. In addition, proteomic analysis showed that zenocutuzumab inhibited the HER3 and AKT-related downstream signaling pathways. In conclusion, these data provide strong mechanistic evidence supporting the clinical efficacy of zenocutuzumab in patients with NRG1 fusion-driven cancers. Citation Format: Jan Gerlach, Igor Odintsov, Ron Schackmann, Marc Ladanyi, Jeroen Lammerts van Bueren, Romel Somwar, Cecile Geuijen. Zenocutuzumab is an effective HER2/HER3 Biclonics® antibody in cancers with NRG1 fusions [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P201.

MiR-665 suppresses the progression of diffuse large B cell lymphoma (DLBCL) through targeting LIM and SH3 protein 1 (LASP1)

Diffuse large B cell lymphoma (DLBCL), the most common type of non-Hodgkin lymphoma worldwide, is aggressive and highly heterogeneous. MiR-665 was found to be lowly expressed in serum exosomes of DLBCL patients and in DLBCL cell lines, but its function in DLBCL progression remains unclear. In this study, miR-665 was overexpressed in SU-DHL-4 cells via miR-665 mimics and knocked down in FARAGE cells via miR-665 inhibitor. Knockdown of miR-665 promoted DLBCL cell proliferation and invasion and decreased cell apoptosis, whereas miR-665 overexpression showed opposite effects on DLBCL cell malignant behaviors. Luciferase reporter assay confirmed LIM and SH3 protein 1 (LASP1) and MYC as target genes of miR-665. Moreover, the expression of LASP1 was negatively correlated with that of miR-665 in LDLBCL cells. LASP1 has tumor-promoting property and its inhibition abolished the effect of miR-665 knockdown on DLBCL cell proliferation, invasion, and apoptosis. SU-DHL-4 cells were inoculated into the flank of nude mice, followed by orthotopic injection with miR-665 agomir. MiR-665 overexpression restricted tumor growth in vivo. Thus, we demonstrates that miR-665 suppresses DLBCL cell malignant behaviors by inhibiting cell proliferation and invasion and inducing apoptosis via targeting LASP1 and MYC, suggesting the importance of miR-665 in DLBCL progression.

Devimistat in Combination with Bendamustine Potently Inhibits Tumor Growth and Promotes CD8+ T Cell Tumor Infiltration in a Syngeneic Mouse Model of CD30+ T-Cell Lymphoma

Introduction: Chimeric antigen receptor-based T cell therapy (CAR-T) has shown great promise in B cell malignancies. CD30-targeted therapies like brentuximab vedotin have activity in T cell malignancies expressing CD30. There is interest in developing CD30-targeted CAR-T therapies for the treatment of relapsed T cell malignancies. As the process of generating autologous T cells takes several weeks, there is a need for active bridging therapeutic regimens that can reduce tumor burden to facilitate subsequent treatment with CAR-T cell therapy. We propose the use of devimistat, a TCA cycle inhibitor, in combination with bendamustine as bridging therapy for the treatment of CD30+ T cell lymphoma (TCL) prior to the use of CAR-T cells. We hypothesize that TCA cycle inhibition will increase the sensitivity of TCL cells to bendamustine, negatively impact the function of immunosuppressive cell populations that depend on mitochondrial metabolism, and create a conducive environment for enhanced CAR-T cell efficacy. To test our hypothesis, we developed a model of CD30+ TCL, evaluated the efficacy of the combination, and characterized its effects by transcriptome profiling of immune cell populations in tumors.Methods: Mouse TCL cell line EL4-LUC2 was engineered to express human CD30 by retroviral transduction. Single and combination in vitro efficacy was evaluated by viability assays. Combination indices (CI) were calculated using Compusyn. CD30+ EL4-LUC2 cells (EL4.CD30) were inoculated subcutaneously into the flanks of C57Bl6 mice, tumor volumes were calculated, and CD30 expression in tumors was evaluated by immunohistochemical staining and flow cytometry. Transcriptome profiling was performed on a representative subset of tumors by single cell RNA sequencing (scRNA-seq).Results: EL4.CD30 cells displayed comparable CD30 expression to the human TCL cell line Karpas 299. EC 50 values for Devimistat and bendamustine in EL4.CD30 cells were 64.9 mM (95% CI: 62.4-67.4) and 101.2 mM (96.2-106.4), respectively; in Karpas 299, EC 50 values for Devimistat and bendamustine were substantially higher: 117.2 mM (114.7-119.6) and 188.2 mM (180.0 - 196.8), respectively. When dosed in combination, devimistat and bendamustine (D/B) showed synergy (CI = 0.5-0.8) at effect levels > 0.9 in 8 of the 16 dose combinations tested. D/B synergy at an effect level > 0.9 was less overt in Karpas 299 with only 2 of 16 tested combination levels displaying CI values of 0.5-0.9. Efficacy studies with EL4.CD30 tumor-bearing mice revealed potent D/B anti-tumor activity relative to vehicle and single-agent treatment groups. Six complete regressions and 4 partial regressions were observed in the D/B group (Fig. 1A). Moreover, tumor growth rates and median survival values were significantly different (p<0.0001) in D/B treated mice (Fig 1B-C). Transcriptome profiling of harvested tumors by scRNA-seq revealed substantial infiltration of cytotoxic T lymphocytes (CTL) exclusively in a combination-treated mouse that responded to therapy (Fig. 2A-C). Direct interrogation of Treg-associated transcripts revealed very low Treg numbers and no clear association to any treatment.Conclusions: Our data demonstrate that D/B has potent pharmacological activity in vitro and in vivo and promotes CTL influx into tumors. This suggests that D/B creates an immunopermissive environment fit for CAR-T cell activity. Subsequent studies will investigate the efficacy of CD30.CAR-T cells following D/B treatment. A Phase II pilot study evaluating the feasibility, safety, and tolerability of D/B in patients with relapsed/refractory T-cell Non-Hodgkin Lymphoma is currently ongoing (NCT04217317). [Display omitted] DisclosuresPardee: Rafael Pharmaceuticals: Consultancy, Research Funding; Karyopharm Pharmaceuticals: Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genetech: Membership on an entity's Board of Directors or advisory committees; BMS: Speakers Bureau; Pharmacyclics: Speakers Bureau.

Acoustic patterning of endothelial cells in vivo to produce microvascular networks

Previous work from our lab developed an ultrasound standing wave field (USWF) technology to noninvasively pattern endothelial cells within collagen hydrogels to fabricate vascularized engineered tissue constructs. Here, we translate this technology towards acoustic patterning and microvascular network formation in vivo. To enable acoustic patterning in vivo, we developed a two-transducer ultrasound system with intersecting beams to produce an USWF at the intersection of the crossed beams. Acoustic radiation forces associated with the USWF patterned cells into planar bands located at nodal planes. Distance between planar bands was controlled through design of acoustic frequency and angle between ultrasound beams. To demonstrate acoustic patterning in vivo, solutions of collagen and human umbilical vein endothelial cells (HUVEC) were injected subcutaneously into flanks of mice and then exposed to an USWF. High-frequency ultrasound imaging was employed to visualize initial acoustic patterning in tissues. Ten days after USWF-mediated cell patterning, tissues were excised, and vessels derived from USWF-patterned cells were identified using an antibody specific to human von Willebrand factor (hVWF). Microvessels expressing hVWF were present in USWF-exposed tissues and not in sham-exposed controls. Furthermore, blood vessels expressing hVWF contained red blood cells, indicating anastomosis of ultrasound-fabricated microvessels with host vasculature and tissue perfusion.

Intratumoral injection of TLR9 agonist promotes an immunopermissive microenvironment transition and causes cooperative antitumor activity in combination with anti-PD1 in pancreatic cancer

BackgroundComplex tumor and immune microenvironment render pancreatic ductal adenocarcinoma (PDAC) resistant to immune checkpoint inhibitors (ICIs). Therefore, a strategy to convert the immune hostile into an immunopermissive tumor is required....

Contribution of nuclear BCL10 expression to tumor progression and poor prognosis of advanced and/or metastatic pancreatic ductal adenocarcinoma by activating NF-\u03baB-related signaling

BackgroundWe previously demonstrated that nuclear BCL10 translocation participates in the instigation of NF-κB in breast cancer and lymphoma cell lines. In this study, we assessed whether nuclear BCL10 translocation is clinically significant in advanced and metastatic pancreatic ductal adenocarcinoma (PDAC).Method and materialsWe analyzed the expression of BCL10-, cell cycle-, and NF-κB- related signaling molecules, and the DNA-binding activity of NF-κB in three PDAC cell lines (mutant KRAS lines: PANC-1 and AsPC-1; wild-type KRAS line: BxPC-3) using BCL10 short hairpin RNA (shBCL10). To assess the anti-tumor effect of BCL10 knockdown in PDAC xenograft model, PANC-1 cells treated with or without shBCL10 transfection were inoculated into the flanks of mice. We assessed the expression patterns of BCL10 and NF-κB in tumor cells in 136 patients with recurrent, advanced, and metastatic PDAC using immunohistochemical staining.ResultsWe revealed that shBCL10 transfection caused cytoplasmic translocation of BCL10 from the nuclei, inhibited cell viability, and enhanced the cytotoxicities of gemcitabine and oxaliplatin in three PDAC cell lines. Inhibition of BCL10 differentially blocked cell cycle progression in PDAC cell lines. Arrest at G1 phase was noted in wild-type KRAS cell lines; and arrest at G2/M phase was noted in mutant KRAS cell lines. Furthermore, shBCL10 transfection downregulated the expression of phospho-CDC2, phospho-CDC25C, Cyclin B1 (PANC-1), Cyclins A, D1, and E, CDK2, and CDK4 (BxPC-3), p-IκBα, nuclear expression of BCL10, BCL3, and NF-κB (p65), and attenuated the NF-κB pathway activation and its downstream molecule, c-Myc, while inhibition of BCL10 upregulated expression of p21, and p27 in both PANC-1 and BxPC-3 cells. In a PANC-1-xenograft mouse model, inhibition of BCL10 expression also attenuated the tumor growth of PDAC. In clinical samples, nuclear BCL10 expression was closely associated with nuclear NF-κB expression (p < 0.001), and patients with nuclear BCL10 expression had the worse median overall survival than those without nuclear BCL10 expression (6.90 months versus 9.53 months, p = 0.019).ConclusionNuclear BCL10 translocation activates NF-κB signaling and contributes to tumor progression and poor prognosis of advanced/metastatic PDAC.

IL-15-induced lymphocytes as adjuvant cellular immunotherapy for gastric cancer.

ObjectivesTo test the antitumor potential of lymphocytes transferred via adoptive cell therapy (ACT) in a mouse model of human gastric cancer (GC), and to evaluate the clinical efficacy and safety of combining lymphocytes as adjuvant therapy with first-line chemotherapy in patients with GC. MethodsWe constructed a human GC xenograft model in sublethally irradiated 6-8-week-old male NCG mice. MKN-45 cells (1 × 106 cells/mouse) were subcutaneously injected into mice's flanks. After tumors had become palpable, we randomized the mice into control, ACTIL-2, and ACTIL-15 groups. Human lymphocytes were then injected into mouse tail veins. In addition, 63 human patients with histologically or cytologically confirmed stage III-IV GC randomly received S-1 + oxaliplatin + ACTIL-15 (combination therapy group) or S-1 + oxaliplatin (chemotherapy group). ResultsIn the mouse study, treatment with ACTIL-15 cells inhibited tumor growth on adoptive transfer, and mice that received ACTIL-15 cells had significantly longer survival rates (p < 0.05, ACTIL-15 vs. ACTIL-2). In the human study, the median survival rate of patients in the combination therapy group was 472days (95% confidence interval [CI], 276-668days), whereas that of patients in the chemotherapy group was 266days (95% CI, 200-332days; p < 0.05). Eleven percent (7/63) of patients had adverse reactions, but these reactions did not interfere with treatment. ConclusionAdoptive transfer of ACTIL-15 cells in a mouse model of GC and in patients with advanced GC treated with S1 + oxaliplatin improved survival rates in both, with an acceptable safety profile.

Tumor Growth in the High Frequency Medulloblastoma Mouse Model Ptch1+/-/Tis21KO Has a Specific Activation Signature of the PI3K/AKT/mTOR Pathway and Is Counteracted by the PI3K Inhibitor MEN1611.

We have previously generated a mouse model (Ptch1+/−/Tis21KO), which displays high frequency spontaneous medulloblastoma, a pediatric tumor of the cerebellum. Early postnatal cerebellar granule cell precursors (GCPs) of this model show, in consequence of the deletion of Tis21, a defect of the Cxcl3-dependent migration. We asked whether this migration defect, which forces GCPs to remain in the proliferative area at the cerebellar surface, would be the only inducer of their high frequency transformation. In this report we show, by further bioinformatic analysis of our microarray data of Ptch1+/−/Tis21KO GCPs, that, in addition to the migration defect, they show activation of the PI3K/AKT/mTOR pathway, as the mRNA levels of several activators of this pathway (e.g., Lars, Rraga, Dgkq, Pdgfd) are up-regulated, while some inhibitors (e.g. Smg1) are down-regulated. No such change is observed in the Ptch1+/− or Tis21KO background alone, indicating a peculiar synergy between these two genotypes. Thus we investigated, by mRNA and protein analysis, the role of PI3K/AKT/mTOR signaling in MBs and in nodules from primary Ptch1+/−/Tis21KO MB allografted in the flanks of immunosuppressed mice. Activation of the PI3K/AKT/mTOR pathway is seen in full-blown Ptch1+/−/Tis21KO MBs, relative to Ptch1+/−/Tis21WT MBs. In Ptch1+/−/Tis21KO MBs we observe that the proliferation of neoplastic GCPs increases while apoptosis decreases, in parallel with hyper-phosphorylation of the mTOR target S6, and, to a lower extent, of AKT. In nodules derived from primary Ptch1+/−/Tis21KO MBs, treatment with MEN1611, a novel PI3K inhibitor, causes a dramatic reduction of tumor growth, inhibiting proliferation and, conversely, increasing apoptosis, also of tumor CD15+ stem cells, responsible for long-term relapses. Additionally, the phosphorylation of AKT, S6 and 4EBP1 was significantly inhibited, indicating inactivation of the PI3K/AKT/mTOR pathway. Thus, PI3K/AKT/mTOR pathway activation contributes to Ptch1+/−/Tis21KO MB development and to high frequency tumorigenesis, observed when the Tis21 gene is down-regulated. MEN1611 could provide a promising therapy for MB, especially for patient with down-regulation of Btg2 (human ortholog of the murine Tis21 gene), which is frequently deregulated in Shh-type MBs.

3: Vitamin D Improves Autologous Fat Graft Retention

Purpose:Autologous fat grafting is a widely used technique in aesthetic and reconstructive surgery, however, unpredictable volume reabsorption may lead to unsatisfactory outcomes. Previously, we demonstrated that a fat-soluble Vitamin D3 analogue, calcitriol, significantly improved fat retention in a xenograft mouse model by 25% across multiple donors when injected systemically (p < 0.05). While calcitriol has minimal toxicity, is FDA approved, and has positive immunomodulatory and antioxidant properties, systemic administration bypasses key Vitamin D synthesis regulatory steps, thus increasing risk with high-dose use. We hypothesize that systemic supplementation with Vitamin D3 (cholecalciferol) will likewise improve fat-graft retention similar to calcitriol while avoiding potential regulatory and iatrogenic risk. In this study we compared in vivo human fat graft retention in mice treated with systemic cholecalciferol, calcitriol or vehicle control in a mouse xenograft model. In vitro adipose lipoaspirate culture was used to interrogate the therapeutic mechanism of action.Methods:Lipoaspirate was harvested from 6 unique donors using a 2mm cannula and used in parallel for both in vitro and in vivo studies. In vivo: 0.3mL of lipoaspirate was injected bilaterally on dorsal flanks of homozygous Foxn1nu immunocompromised mice. Calcitriol (50ng), cholecalciferol (50ng, 500ng, 5000ng) or vehicle control was administered thrice weekly by IP injection. Graft volume retention was measured at 12 weeks. In vitro: 1mL of lipoaspirate was submerged in phenol-red free DMEM (10% FBS) containing calcitriol or cholecalciferol (15.6nM, 62.5 nM or 250 nM) for 7 days with one media change. Terminal analyses include tissue weight, stromal cell viability, concentration of active vitamin D metabolite (1,25(OH)2D3), and gene upregulation by qRT-PCR.Results:Previously, we demonstrated that systemic administration of 50ng calcitriol thrice weekly significantly improved human fat graft retention across multiple donors in a mouse xenograft model. Our current in vivo data suggest 5000ng cholecalciferol is similarly effective. In-vitro assays show 62.5nM and 250nM cholecalciferol significantly increased adipose stromal cell viability compared to controls (85.3+/-2.9% and 87.7+/-3.7 versus 77.6+/-2.8%, respectively p<0.05;). Analysis of final adipose 1,25(OH)2D3 concentration by ELISA showed both calcitriol and cholecalciferol treatments equally increased Vitamin D metabolite concentrations in all donors. qRT-PCR analysis of gene expression show that pro-survival autophagy is significantly increased by both cholecalciferol and calcitriol, though increased concentration of calcitriol was required to induce significant increases from controls.Conclusion:Cholecalciferol (Vitamin D3) is a highly promising therapeutic for improving fat grafting outcomes. Our in vitro data suggests that in the context of hypoxia, nutrient depletion, or growth factor deprivation, such as occurs immediately following fat grafting, vitamin D3 promotes stromal cell autophagy. In this context, autophagy is crucial for maintaining cellular ATP production and macromolecular synthesis and, therefore, represents an essential pro-survival pathway which allows grafted cells to survive. The results herein provide evidence to incorporate vitamin D3 as a safe, cost-effective nutritional supplement into the perioperative workflow to improve fat viability after grafting.

Antitumor effects of AZD2014, a dual mTORC1/2 inhibitor, against human hepatocellular carcinoma xenograft in nude mice

To investigate the antitumor effects of AZD2014 (a dual mTORC1/2 inhibitor) against human hepatocellular carcinoma (HCC) xenograft in mice. HCCLM3 cells were injected subcutaneously in the right flank of nude mice, and when the tumors were macroscopic, the mice were randomized into 2 groups for daily intraperitoneal injection of AZD2014 (5 mg/kg, n=5) or vehicle (5 mL/kg, n=5) for 24 days. Tumor growth was assessed using calipers every 4 days and the tumor growth curve was drawn. After the final injection, the mice were euthanized and the tumors were dissected for measuring tumor weight and histopathological analysis with HE staining. Immunohistochemical staining was used to detect the expressions of Ki-67, cleaved caspase-3, CD31, and the epithelial-mesenchymal transition (EMT)-related proteins (Ecadherin, N-cadherin, and vimentin) in the tumor tissue. Daily treatment with AZD2014 significantly suppressed HCC growth as compared with the control group. HE staining showed significantly increased tumor necrosis in AZD2014-treated mice. AZD2014 treatment inhibited tumor cell proliferation, angiogenesis and EMT progression as shown by decreased expressions of Ki-67, CD31, N-cadherin, and vimentin and increased expression of E-cadherin in the tumor tissue, and significantly promoted tumor cell apoptosis as shown by an increased expression of cleaved caspase-3 in AZD2014-treated mice. AZD2014 is a highly potent antitumor agent for HCC in nude mice bearing HCC xenografts. AZD2014 can effectively inhibit tumor proliferation, angiogenesis and EMT progression and induce tumor cell necrosis and apoptosis.

Irradiation of subcutaneous mouse tumors with a clinical linear accelerator validated by alanine dosimetry

Preclinical radiotherapy studies using mouse models are an indispensable step on the path from in vitro experiments to clinical implementation. In support of this step, dedicated small animal irradiators have been developed to provide a platform for testing new radiation strategies for cancer treatment. Although some systems offer an advanced setup including high resolution positioning and imaging, the investment in obtaining, maintaining, and using small animal irradiators is substantial both monetary and in man-hours. An alternative approach is to use linear accelerators (LINACs) designed for irradiation of humans and widely available in clinical environments, as they may potentially increase quality and translational value of preclinical cancer research.Here we present a simple setup for fast and localized irradiation of subcutaneous mouse flank tumors using a clinical 10 MV flattening filter free (FFF) LINAC beam. Alanine pellet dosimeters were used for validation of the setup. Good agreement was found between alanine and the vertical Eclipse dose profile both outside and inside the field. Additionally, the alanine pellets verified the in vivo dose to the mouse tumor location by comparing and finding agreement between the dose from ten subcutaneously inserted dosimeters and ten externally placed dosimeters. Finally, the setup was tested using the syngeneic CT26 colon cancer mouse model. A single dose of 15 Gy was provided to subcutaneous tumors on the right flank. Comparison of tumor growth between the irradiated and non-irradiated tumors showed a delay in tumor growth and improved overall survival amongst the irradiated mice.This study demonstrates that clinical LINACs are an easy, fast, and cost effective alternative to small animal irradiators without compromising accurate dose delivery. Clinical LINACs could be utilized to a greater extent in preclinical studies.

Pharmacological inhibitors of anaplastic lymphoma kinase (ALK) induce immunogenic cell death through on-target effects

Immunogenic cell death (ICD) is clinically relevant because cytotoxicants that kill malignant cells via ICD elicit anticancer immune responses that prolong the effects of chemotherapies beyond treatment discontinuation. ICD is characterized by a series of stereotyped changes that increase the immunogenicity of dying cells: exposure of calreticulin on the cell surface, release of ATP and high mobility group box 1 protein, as well as a type I interferon response. Here, we examined the possibility that inhibition of an oncogenic kinase, anaplastic lymphoma kinase (ALK), might trigger ICD in anaplastic large cell lymphoma (ALCL) in which ALK is activated due to a chromosomal translocation. Multiple lines of evidence plead in favor of specific ICD-inducing effects of crizotinib and ceritinib in ALK-dependent ALCL: (i) they induce ICD stigmata at pharmacologically relevant, low concentrations; (ii) can be mimicked in their ICD-inducing effects by ALK knockdown; (iii) lose their effects in the context of resistance-conferring ALK mutants; (iv) ICD-inducing effects are mimicked by inhibition of the signal transduction pathways operating downstream of ALK. When ceritinib-treated murine ALK-expressing ALCL cells were inoculated into the left flank of immunocompetent syngeneic mice, they induced an immune response that slowed down the growth of live ALCL cells implanted in the right flank. Although ceritinib induced a transient shrinkage of tumors in lymphoma-bearing mice, irrespective of their immunocompetence, relapses occurred more frequently in the context of immunodeficiency, reducing the effects of ceritinib on survival by approximately 50%. Complete cure only occurred in immunocompetent mice and conferred protection to rechallenge with the same ALK-expressing lymphoma but not with another unrelated lymphoma. Moreover, immunotherapy with PD-1 blockade tended to increase cure rates. Altogether, these results support the contention that specific ALK inhibition stimulates the immune system by inducing ICD in ALK-positive ALCL.

Abstract 2790: Temporal immune changes of murine breast and melanoma tumor microenvironments following pulsed focused ultrasound

Abstract Various cellular and biological immunotherapies have revolutionized the treatment of human malignancies by augmenting endogenous immune responses within the tumor microenvironment (TME). Although non-ablative pulsed-FUS (pFUS) has received less attention, it has been successfully used as a noninvasive treatment of solid malignancies by stimulating a pro-inflammatory immune response within the TME. Such an approach could be useful to treat peripheral micro-metastases in normal-appearing tissues. In the present study, we interrogated the immune-modulatory changes that occur within the TME following pFUS exposure. Murine B16 melanoma and 4T1 mammary carcinoma cells were subcutaneously implanted into both mice flanks (n=10 tumors/group/time point). The natural histories cytokine, chemokine, trophic factor (CCTF), and cell adhesion molecule (CAM) expression was evaluated within the TME of untreated flank tumors over 11 days. Ultrasound-guided pFUS at 1 MHz and peak negative pressures of 6 MPa was administered to tumors after reaching ~5 mm in diameter. Temporal immune-related changes in tumors were assessed following sonication using using proteomic, transcriptomic and histological analyses. Natural history growth characteristics showed a progressive increase in size for both tumors, and proteomic analysis revealed a shift toward an immunosuppressive TME. An anti-tumor TME was detected following pFUS treatment with increased expression of various pro-inflammatory CCTF and CAM as well as reduced tumor growth rates. Transcriptomic analyses following sonication identified inverted gene expression patterns in the two tumor types. Functional analysis of B16 tumors revealed increased intracellular signaling pathways associated with immune response regulations, while 4T1 tumors demonstrated reduced expression of proliferation genes. This study provides a macroscopic overview on the temporal dynamics of the TME and underline the profound magnitude of tumoral heterogeneity between tumor types as well as through progressive stages of similar primary tumors. Nevertheless, the induced immune-modulation changes suggest that pFUS may alter the expression of pro-inflammatory CCTF, CAM, towards an anti-tumoral TME, and supports the potential use of pFUS as a neoadjuvant treatment approach. Citation Format: Gadi Cohen, Parwathy Chandran, Rebecca M. Lorsung, Lauren E. Tomlinson, Robert B. Rosenblatt, Scott R. Burks, Joseph A. Frank. Temporal immune changes of murine breast and melanoma tumor microenvironments following pulsed focused ultrasound [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2790.

Abstract 70: Obesity-induced impairment of antitumor immunity is associated with an immunosuppressive tumor immune landscape

Abstract Obesity is associated with an increased risk and progression in a variety of cancers. In melanoma, obesity is associated with an increased risk of recurrence following surgical resection which may reflect immune escape of cancer surveillance. We sought to examine the impact of obesity on antitumor immunity in an immunogenic preclinical melanoma model.Male C57/Bl6 mice were fed a 45% fat diet or 10% fat diet for 12 months resulting in obese (body weight 52.8g ±1.3) vs control mice (body weight 38.8g ±2.2). For these studies, we utilized the YUMMER cell line, a C57BL/6J syngeneic melanoma cell line with three driver mutation/deletions (BrafV600E, Pten-/-, Cdkn2a-/-), irradiated to mimic the high mutation burden of human melanoma. 0.5x106 YUMMER cells were subcutaneously injected into the flank of aged (13 month old) control and obese mice. After 14 days post injection, tumors in control mice either had growth arrest or regression. In contrast, all obese mice exhibited progressively growing tumors with 6-fold greater tumor weights after 21 days as compared to control (1.3g ±0.4 vs 0.23g ±0.06). CyTOF analysis of tumor-infiltrating immune cells at day 21 showed 2-fold decrease in the percentage of CD4 T cells in tumors from obese mice (9.8% vs 21.1% of CD45). In addition, obesity resulted in 4-fold increase in the frequency of intratumoral Treg (26.1% vs 8.3% of CD3CD4 cells) and CD8/Treg ratio was 2-fold reduced in obese mice. In contrast to prior findings in obese mice bearing B16 melanomas, the percentage of CD8 T cells expressing PD1 was similar (80.4% vs 74.3%), but we found 2-fold higher frequency of CD8PD1 T cells co-expressing TIM3 and LAG3 in tumors from obese mice (43.3% vs 23.0%). Moreover, obesity also attenuated the frequency of tumor-infiltrating cDC2 (1.4% vs 3.5% of CD45) but did not affect NK cells or B cell proportions. Furthermore, the ratio of tumor infiltrating myeloid cells to T cells was higher in obese mice (1.94:1 vs 0.82:1). Moreover, myeloid cells from obese mouse tumors had higher expression of PDL1, CD206, and CD38 markers.Our findings provide evidence that obesity impairs antitumor immunity by modulating the relative abundance of intratumoral immune cell populations and their phenotypes to promote immunosuppressive microenvironment. These findings will be validated in subsequent studies and examined in human specimens. Citation Format: Barbara Pazdrak, Duncan Mak, William Damsky, Marcus Bosenberg, Brooklyn Lochmann, Matthew Gubin, Jennifer McQuade. Obesity-induced impairment of antitumor immunity is associated with an immunosuppressive tumor immune landscape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 70.

Abstract 2710: The MD Anderson patient-derived xenograft series for modeling precision oncology in biliary tract cancers

Abstract Purpose: Biliary tract cancers (BTCs), including intrahepatic cholangiocarcinoma (ICC), extrahepatic cholangiocarcinoma (ECC), and gallbladder cancer (GBC), are rare malignancies which frequently present at advanced stage and have a poor survival. Incorporation of next-generation sequencing into clinical care has provided an avenue for the development of targeted therapies in BTCs which are rich in actionable mutations. However, clinically relevant models for BTC are limited, representing a major challenge in the field. We sought to develop a catalog of BTC patient-derived xenograft (PDX) models and representing diverse molecular profiles to create a resource for the modeling of precision oncology. Methods: Tumor tissue from surgical specimens or image-guided biopsies was collected from consenting patients at MD Anderson Cancer Center with histologically-confirmed BTCs for PDX development. Tumor fragments were implanted in the flank of NSG mice and passaged into nude mice. Whole-exome sequencing (WES) was performed on tumors from early-passage (P1-P2) PDXs and matching patient blood to determine somatic mutations/indels and copy number variations. Functional alterations in cancer-related genes that are targetable directly or indirectly with approved or investigational agents were considered “actionable”. Results: Samples were obtained from 97 patients undergoing image-guided biopsy (86/97, 89%) or surgical resection (11/97, 11%). Out of 97 tumors implanted, 33 (34%) PDXs from 30 patients were successfully established: 74% (24/33) ICC, 15% (5/33) ECC, and 12% (4/33) GBC. Relative take rates for PDX development were 34% (29/86) for those developed from biopsy samples and 36% (4/11) for those developed from surgical samples. In two patients, multiple PDXs were developed from biopsies at different time points during their care. WES demonstrated that the PDXs captured several key actionable alterations including alterations in ERRB2, FGFR2, IDH1, ATM, PIK3CA, PTEN and KRAS. Conclusion: Here, we describe a unique collection of clinically and molecularly annotated BTC PDX models which reflect the genomic heterogeneity present in ICC, ECC, and GBC. WES of early-passage PDXs provides evidence that these models capture the variety of actionable alterations harbored in the parental tumor and may enable the development of novel, biomarker-driven treatment strategies and rational combination therapies. To date, this is one of the largest collections of BTC PDX models available and will serve as an invaluable resource to guide the development of personalized treatments for patients with these aggressive malignancies. Citation Format: Timothy Philip DiPeri, Kurt W. Evans, Ching-Wei D. Tzeng, Lawrence Kwong, Michael P. Kahle, Xiaofeng Zheng, Dali Li, Hop S. Tran Cao, Thuy Vu, Sunhee Kim, Fei Su, Bryce Kirby, Chetna Wathoo, Gabriela Raso, Yasmin Rizvi, Huamin Wang, Filip Janku, Kenna Shaw, Timothy Yap, Milind Javle, Jordi Rodon, Funda Meric-Bernstam. The MD Anderson patient-derived xenograft series for modeling precision oncology in biliary tract cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2710.

Abstract 1388: ATM inhibitor AZD1390 sensitizes breast cancer brain metastasis to radiation therapy

Abstract Metastasis to the central nervous system (CNS) has remained a major source of morbidity and mortality for patients with breast cancer. This is mainly driven by the lack of therapeutic options, as traditional chemotherapy and targeted therapies have been shown to have limited efficacy in the brain. Ataxia-telangiectasia mutated (ATM) protein orchestrates the cellular DNA damage response (DDR) to cytotoxic DNA double-strand breaks induced by ionizing radiation (IR). ATM genetic ablation or pharmacological inhibition results in tumor cell hypersensitivity to IR.AZD1390 is a potent orally bioavailable ATM inhibitor, specifically optimized for blood brain barrier penetration and has shown to be an effective radiosensitizer in gliomas. We have shown that there are frequent alterations in DNA damage response (DDR) pathways in CNS metastasis (CM), which include frequent mutations in TP53. Incorrect repair of DNA lesions often leads to genomic instability. ATM, a core component of the DNA repair system, is activated to enhance the homologous recombination (HR) repair pathway upon DNA double-strand breaks. Here we show that AZD1390, when used in combination with radiation therapy, can inhibit the growth of breast cancer CM in patient derived xenograft (PDX) tumors. Three PDX models (2 Her2+ and one triple negative), all of which harbor TP53 mutations and other DDR alterations, were implanted into the flank or brain of immunocompromised mice. Pre-treatment with AZD1390 followed by a fractionated regime of radiation therapy (2Gy/day for 5 days) significantly inhibited growth of flank PDX tumors by an average of 81% (85% and 80% in the HER2+ and 77% in the triple negative PDX, p-value &amp;lt; 0.05). This is compared to an average of 7% (8%, 13% and 0% respectively) for tumors treated with AZD1390 alone or 40% (39%, 21% and 58% respectively) for radiation therapy alone in all PDX models. Growth inhibition was sustained until the experiment endpoint at 90 days in 75% of the tumors, and 25% of the tumors regressed below their size at the beginning of treatment. About 31% of tumors across three models started to grow back and gene expression analysis comparing these tumors with complete responders demonstrated changes in oxidative phosphorylation, sirtuin signaling, and ERK/MAPK signaling. Adjuvant therapy of AZD1390 did not further decrease tumor growth. Our results demonstrate that AZD1390 can sensitize breast cancer CM with DDR pathway alterations to radiation therapy. This points to the potential of AZD1390 as a potential novel therapeutic agent for patients with breast cancer CM. Citation Format: Ben Yi Tew, Stephen Durant, Bodour Salhia. ATM inhibitor AZD1390 sensitizes breast cancer brain metastasis to radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1388.

Abstract 2339: Halting head &amp; neck squamous cell carcinoma progression by broadly targeting glutamine metabolic pathways

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, resulting in over 14,600 deaths each year in the United States alone. HNSCC is associated with human papillomavirus (HPV) infection, and tobacco use and abusive alcohol intake. Recent revolutionary immunotherapy approaches have changed the landscape of treatment options in HNSCC. However, less than 20% of HNSCC patients respond to FDA-approved anti-PD1 immune checkpoint blockade (ICB) (pembrolizumab and nivolumab), often not leading to durable responses. This highlights the unmet need to identify novel therapeutic options and biomarkers predicting more favorable response to maximize the efficacy of immune-oncology (IO) strategies for HNSCC treatment. Glutamine is a conditionally essential amino acid for rapidly proliferating cancer cells making glutamine pathway inhibition an attractive approach for anti-cancer therapy. Treatment with the glutamine antagonist (DRP-104), which irreversibly inhibits all known enzymes involved in glutamine metabolism within the tumor, resulted in metabolically halted cell growth in vitro in a large panel of cell lines (n=8) representing the spectra of HPV- and HPV+ HNSCC, with an IC50 of 0.2-25uM, Interestingly, HNSCC cells bearing genetic alterations in PIK3CA/PTEN were significantly more sensitive to glutamine antagonism in comparison to unaltered HNSCC cells. The dependence of glutamine in HNSCC growth and the increased sensitivity of PIK3CA/PTEN aberrant cells was also observed in orosphere assays and 6 distinct HNSCC tumor xenografts in NOD SCID mice. We next investigated the efficacy of glutamine suppression in the context of an intact immune system, taking advantage of new syngeneic HNSCC model system, 4MOSC and MOC1. Specifically, we recently developed orthotopic syngeneic mouse models, 4MOSC, reflecting human tobacco-related HNSCC genomic alterations and mutational landscape (Wang et al., Nature Comm 2019), while MOC1 is a DMBA-induced mouse HNSCC cell line grown in the subcutaneous flank of mice (Judd et al. Cancer Res 2011). Monotherapy administration of DRP- 104 (1.4mg/kg) was potent as a monotherapy in all three syngeneic HNSCC models. We also observed a trend of increased sensitivity when DRP-104 was combined with anti-PD-1 therapy. Our data suggest that broad glutamine antagonism using DRP-104 has therapeutic potential in HNSCC by both dismantling cancer metabolism and enhancing the anti-tumor immune response of immune checkpoint blockade. Clinical trials of DRP-104 are currently ongoing. Citation Format: Michael Allevato, Yumi Yokoyama, Robert Wild, Silvio Gutkind. Halting head &amp; neck squamous cell carcinoma progression by broadly targeting glutamine metabolic pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2339.

Abstract LB249: Development of 3D organoid raft cultures of colon cancer as a model to screen the therapeutic efficacy of PRIMA-1Met (APR-246)

Abstract Background: Cancer is among the leading causes of death worldwide. Hence, new drugs and faster screening models are needed. However, costly experiments for determining the therapeutic efficacy of drugs in animals before they reach clinical trials make the process difficult. To address this issue, we developed an alternative strategy to screen drugs against human tumors. 3D tumor organoids are termed Organoid Raft Cultures (ORCs) due to the strategy involved in culturing tumors ex vivo on collagen beds with grid supports to maintain their morphological structure and molecular makeup. Methodology: After receiving informed consent of patients and IRB approval at the University of Alabama at Birmingham, patients with histologically confirmed colon cancers were recruited. Portions of collected colon tumors were then implanted into the flanks of mice to generate Patient-Derived Xenografts (PDXs). Once their volumes reached 2000 mm2, tumors were excised, and ORCs were generated. This approach addresses two concerns: 1) how well are the morphological features retained when the tumors are grown ex vivo, and 2): can we accomplish faster screening of the efficacy of drugs. In the present study, we used a tumor in which we identified, by DNA-sequencing, a TP53 missense mutation at amino acid R248Q (a change from arginine to glutamine). We tested PRIMA-1Met (APR-246), an agent that reactivates mutant p53 by causing a conformational change and restores its function as a tumor suppressor. Excised xenograft tumor tissue was cut into small pieces and seeded on mucosa-stromal equivalent consisting of buffered collagen and fibroblasts in 24-well plates and allowed to grow for 2-3 days. Next, the tissue assembly was detached and placed on raft shaped stainless steel grids in 6-well plates containing ORC media. These cultures, termed ORCs, were then divided into groups of APR-246 treated and untreated. After 7 days, tissues were formalin fixed, and slides of 5-micron thickness were cut for Hematoxylin and eosin (H&amp;E), p53 and TUNEL staining. Results: H&amp;E staining of PDXs and ORCs showed the normal glandular morphology of colon cancers. This conventional characteristic of adenocarcinoma was retained along with other morphologic features, indicating the usefulness of ORCs. Additionally, comparisons of p53 and TUNEL staining of untreated vs APR-246-treated ORCs showed that lack of nuclear accumulation of mutant p53 in this p53 mutant tissue and induced apoptosis by APR-246 treatment, indicating the efficacy of the drug and the usefulness of the ORCs as alternative screening model. Conclusion: ORCs are economical and will be useful for screening new drugs/therapies ex vivo, allowing faster drug screening and thus moving new agents to clinical trials. This work was supported in part by the ELKUS foundation. (*Equal contributing first coauthors) Citation Format: Sanjib Nilam Banerjee, Prachi Bajpai, Amr Elkholy, Dianne W. Moore, Hyung Gyoon Kim, Sumit Agarwal, Michael Behring, Sameer Al Diffalha, Upender Manne. Development of 3D organoid raft cultures of colon cancer as a model to screen the therapeutic efficacy of PRIMA-1Met (APR-246) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB249.

Deletion of Calr By CRISPR/Cas9 Genome-Editing Mimics Abnormalities of Megakaryocytopoiesis Induced By Calr Del52 Mutation

INTRODUCTION. Calreticulin (CALR) is mutated in 20-25% of patients with myeloproliferative neoplasms (MPN). Most common abnormalities are deletion of 52bp (T1) and insertion of 5bp (T2) in exon 9, causing recurrent frameshift and generation of novel C-terminal domain. Interactions of mutant CALR with the thrombopoietin receptor MPL contribute to abnormal megakaryocytopoiesis of MPN (Chachoua I, Blood 2016; Araki M, Blood 2016; Elf S, Cancer Discov 2016), but pathogenic mechanisms remain largely unknown.METHODS. Rabbit antibodies (pAb) against mutated CALR were obtained and extensively characterized for their binding to the mutated CALR only. CALR knock-out (KO) and CALR-T1 variants were generated by CRISPR/Cas9 technology in K562 cells; transient expression of CALR-WT, T1 and T2 was obtained by transfecting KO cells. UT7 cells stably expressing CALR-T1, T2 and WT were generous gift of W. Vainchenker. For in-vivo experiments, nude mice were injected subcutaneously with K562 CALR-KO, WT, T1 and T2 cells and tumor mass was measured.RESULTS. We found that genome-edited K562 CALR-T1 cells expressed similar levels of WT and del52 CALR transcripts (by qRT-PCR) while no amplification was obtained in KO cells; correctly edited sequence in T1 cells was assessed by Sanger. Presence of both WT and T1 CALR protein with the predicted molecular weight was confirmed by immunoblotting; no immunoreactivity was found in KO lysates. Interestingly, we identified a cleaved CALR product (about 40KDa) in K562 CALR-T1 and UT7 CALR-T1/T2 cells, whose generation was inhibited by 15mM ROS-inhibitor N-acethyl-L-cysteine (NAC), suggesting that the mutated protein is involved in oxidative stress pathway. By confocal microscopy with mutation-specific antibodies, the mutant CALR was localized in both cytosolic compartment and cell membrane of K562 CALR-T1 and UT7 CALR-T1 and -T2 cells. No significant differences in cell cycle and proliferative rate of K562 KO and T1 cells were observed in comparison to parental cells. However, K562 KO and T1 cells were more resistant to apoptosis compared with parental cells (p<0.05). When induced to Mk commitment in liquid cultures with phorbol-myristate-acetate (PMA), KO and T1 cells both showed accelerated and enhanced expression of CD41/CD61 vs parental cells (2.3- and 3-fold higher, respectively; p<0.01). These data were further confirmed in K562 KO and KO cells overexpressing CALR-WT, T1 and T2, where the proportion of CD41/CD61 positive cells raised from 30% in WT cells to 55%, 62%, 70% respectively in T1, T2 and KO cells (p<0.01). We also produced CRISPR/Cas9 CALR-KO CD34+ from cord blood; clonogenic assays showed that CALR-KO CD34+ cells generated CFU-Mk and BFU-E colonies 8- and 2.5-fold more, respectively, than untouched cells (n=5; p<0.05). Overall, these data indicated that deletion of CALR by genome editing resulted in promotion of megakaryocytopoiesis, and to less extent of erythropoieisis, in normal CD34+ cells, mimicking the effects obtained in K562 CALR-KO and T1 induced to Mk differentiation by PMA.A number of differential expressed mRNAs was found by target profiling in K562 KO and T1 cells. One recurrently affected gene category was “integrin-binding and adhesion molecules”; in particular, ICAM-1, Osteonectin and Stathmin, involved in integrin and collagen binding and cytoskeleton regulation, were significant down-regulated (-40%, -50%, -60% respectively, vs parental cells; p<0.05). Clarification of their role in normal megakaryocytopoiesis is ongoing. The functional relevance of those deregulated genes was supported by in-vivo experiments, where K562 CALR-WT, KO or T1 cells were injected subcutaneously in different flanks of nude mice. We found that K562 KO and T1 cells were both unable to generate a measurable tumor mass while WT cells rapidly produced visible tumors up to 2g weight 20 days post-injection, suggesting defective anchorage-mediated tumor growth. These findings are in line with reports indicating inability of CALR-KO solid tumor cells to engraft (Feng R, Diagn Pathol, 2015; Feng S, Clin Cancer Res, 2014).CONCLUSIONS. These results suggest that loss of wild-type CALR by CRISPR/Cas9-mediated target deletion in cell lines and primary CD34+ cells phenocopies the effects of CALR del52 mutations on megakaryocytopoiesis. DisclosuresVannucchi:Shire: Speakers Bureau; Novartis: Honoraria, Speakers Bureau.