Primary Plasma Cells Expressing CD52 Are Efficiently Targeted In Vivo by Alemtuzumab.

Abstract Salvage therapy options for multiple myeloma (MM) patients relapsing following autologous stem cell transplantation remain limited. New treatments targeting the malignant plasma cell (PCs) are therefore needed. Alemtuzumab is a humanized monoclonal antibody to CD52 capable of destroying CD52+ cells by antibody-mediated cellular cytotoxicity and complement fixation. In order to evaluate the therapeutic potential of alemtuzumab for MM patients, we initially examined CD52 expression on primary malignant marrow PCs as well as a panel of MM continuous cell lines (RPMI-8226, KMS-11, OPM-2, U-266, LP-1, ARH-77). In addition, the anti-myeloma activity of alemtuzumab was evaluated in vivo in a xenotransplant model of MM in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. PCs were enriched from the marrow of MM patients (n = 40) according to CD138 expression. By using an immunomagnetic technique (Miltenyi Biotec, Germany, EU), highly purified CD138+ cells (median purity = 93%; median recovery = 55%) were obtained and further characterized by 3-color (CD138/CD45/CD52) flow cytometry. Expression of CD52 on CD138-enriched cells was detected in 28/40 (70%) of MM patients, with a median of 95% PCs expressing CD52. Detection of CD52 was equally evident on CD138+CD45+ and CD138+CD45− PCs (P ≥ 0.05). Similarly to what observed for primary CD138+ cells, MM cell lines showed a rather heterogeneous CD52 expression, ranging from dim (KMS-11, OPM-2) to bright positivity (LP-1, ARH-77). Both on primary PCs and MM cell lines, expression of CD52 mRNA by quantitative PCR analysis strongly correlated with CD52 antigen detection by flow cytometry. The in vivo activity of alemtuzumab was evaluated in a xenotransplant model of MM in NOD/SCID mice. Mice were inoculated intravenously with KMS-11 cells (0.5 x 105 per mice) and were treated with alemtuzumab (3 x 1 mg/mouse, subcutaneously, days 2, 5, 7). All placebo-treated mice (n = 15) died, whereas mice treated with alemtuzumab (n = 15) showed a significant prolongation of median survival (P ≤0.009 by log rank test) and 27% of them were alive and well at 120 days. No mice experienced any apparent treatment-related toxicity. According to these data, we conclude that: (1) CD52 is expressed on the plasma cells of a significant proportion of MM patients; (2) alemtuzumab has a strong antitumor activity in vivo on CD52-positive MM cell lines; (3) alemtuzumab might have therapeutic potential in a subset of MM patients.

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FLT3 Immunotherapy Can Eliminate Engraftment of ALL Cells in NOD/SCID Mice.

Blood ◽

10.1182/blood.v106.11.869.869 ◽

2005 ◽

Vol 106 (11) ◽

pp. 869-869

Author(s):

Obdulio Piloto ◽

Bao Nguyen ◽

Patrick Brown ◽

Kyu-Tae Kim ◽

David Huso ◽

...

Keyword(s):

Cell Lines ◽

Scid Mice ◽

Cytotoxic Agents ◽

Radioactive Isotopes ◽

Leukemic Cells ◽

Pcr Analysis ◽

Facs Analysis ◽

Antibody Treatment

Abstract The class III receptor tyrosine kinase, FLT3, is expressed by over 90% of B-lineage acute lymphoblastic leukemias (ALL) blasts. In addition, it is expressed at extremely high levels in ALL patients with MLL-rearrangements or hyperdiploidy and sometimes mutated in these same patients. In this report, we investigated the effects of EB10, an anti-human FLT3 monoclonal antibody capable of preventing binding of FLT3 ligand (FL), on ALL cell lines and primary cells. In vitro studies, examining the ability of EB10 to inhibit FLT3 activation and downstream signaling in ALL cell lines and primary blasts, yielded variable results. In some cell lines FLT3 phosphorylation was inhibited and with it, downstream activation of pathways involving MAPK, AKT, and STAT5 phosphorylation. However, several cell lines actually exhibited FLT3 activation upon antibody treatment, possibly because of antibody-mediated receptor dimerization, and subsequent activation of downstream pathways. Nevertheless, through antibody-mediated cellular cytotoxicity (ADCC) such an antibody could still prove efficacious against leukemia cells in vivo. In fact, EB10 treatment significantly prolongs survival and/or reduces engraftment of several ALL cell lines and some primary ALL samples in NOD/SCID mice, even when EB10 treatment results in FLT3 activation of those cell lines in vitro. Moreover, FACS and PCR analysis of EB10 treated NOD/SCID mice surviving 150 days post leukemic cell injection revealed that FLT3 immunotherapy eliminated leukemic engraftment. The leukemic cells surviving EB10 treatment in the mice were characterized by FACS analysis and found to express lower levels of FLT3. To assess for resistance, cells surviving EB10 treatment were injected into NOD/SCID mice and treated with a single dose of EB10. FACS analysis revealed that these cells remain sensitive to EB10 treatment. Taken together, these data demonstrate that EB10 is cytotoxic to ALL blasts in vivo and EB10 treatment did not select for resistant clones. Such an antibody, either naked or conjugated to radioactive isotopes or cytotoxic agents, may prove useful in the therapy of infant ALL as well as childhood and adult ALL patients whose blasts typically express FLT3.

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siRNA for the Ig Light Chain Constant Region Reduces Light Chain Production and Secretion By Human Plasma Cells and in a Murine Xenograft Model

Blood ◽

10.1182/blood.v124.21.5722.5722 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5722-5722

Author(s):

Xun Ma ◽

Ping Zhou ◽

Monika Pilichowska ◽

Chakra P Chaulagain ◽

Sandy Wong ◽

...

Keyword(s):

Flow Cytometry ◽

Human Plasma ◽

Cell Lines ◽

Light Chain ◽

Plasma Cells ◽

Xenograft Model ◽

Myeloma Cell ◽

Constant Region

Abstract Background Ig light chain (LC) diseases such as AL amyloidosis and monoclonal light-chain deposition disease are caused by pathologic free LC. Treatment is aimed at eliminating LC production but success is limited. RNA interference (RNAi) can stop LC production but the diversity of LC variable region sequences poses a challenge that targeting consensus sequences in the constant region (CR) of LC mRNA may overcome (Blood 2014;123:3440). We have developed siRNA pools designed to target the κ or λ LC CR mRNA in human plasma cells and impair LC production and secretion, and have shown that the pool targeting the λ LC CR can do so, and can also trigger a terminal unfolded protein response in clones producing intact Ig due to intracellular accumulation of unpaired heavy chains (ibid). Here we report the results of continued in vitro and in vivo testing of these pools in patient specimens and in a murine xenograft model. Methods Pools of siRNA for the κ or λ LC CR (si[IGLCκCR], si[IGLCλCR]) were custom produced with a non-target control (si[-]). They were introduced in vitro into human plasma cells by an optimized streptolysin O-based method (SLO) and in a NOD.SCID xenograft flank plasmacytoma model by in vivo electroporation as per Gene Therapy 2011;18:1150. In vitro we evaluated LC gene expression, production and secretion at 24 hours in human myeloma cell lines and CD138-selected specimens from patients with plasma cell neoplasms, using real-time PCR (qPCR) for LC mRNA, flow cytometry for intracellular LC mean fluorescence intensity (MFI) and ELISA (Bethyl Laboratories) for LC secretion in 24-hour suspension cultures (106 cells/ml). In vivo we inoculated each of the flanks of NOD.SCID mice with 107 human myeloma cells (ALMC-1 or ALMC-2). When plasmacytomas were 0.5cm3 we injected si[IGLCλCR] or si[-] one time to each flank plasmacytoma respectively, allowing each mouse to serve as its own control. Two days later, the mice were sacrificed and the plasmacytomas excised for qPCR for λ LC mRNA and serum was obtained to measure human λ LC levels by ELISA. Results We have previously described results with siRNA targeting the λ LC CR in human cell lines that make λ LC (ALMC-1, ALMC-2, EJM, OPM2, MM.1S, and MM.1R) and in 16 AL λ patient specimens. We demonstrated significant decreases in LC mRNA, intracellular LC MFI, and λ LC secretion by cell lines (Blood 2014;123:3220); moreover, transcriptional profiling indicated minimal off-target effects (ibid; Supplement). We now report that in vitro secretion of λ LC by CD138-selected plasma cells from AL patients (n=3, newly diagnosed λ) treated with si[IGLCλCR] was reduced by 65% from a mean of 3.1 to 1.0µg/ml and that the residual λ LC mRNA was 49% of control. Similarly we treated κ LC secreting human myeloma cell lines with si[IGLCκCR] and si[-] (IM9, H929, JJN-3, and ARH77). By qPCR the residual κ LC mRNA was 13%, by flow cytometry the MFI was reduced by a median of 67.3% (22.5-90.8), and by ELISA mean κ LC secretion was reduced from 3.7 to 0.8µg/ml (P = 0.055, paired t test). We treated CD138-selected κ patient samples (AL 3, LCDD 1, MM 6) in the same way. By qPCR the residual κ LC mRNA was 57% control, by flow cytometry the MFI was reduced by a median of 37.5% (14-69.8), and by ELISA secretion was reduced from 9.4 to 6.5µg/ml (P = 0.02, paired t test). In the murine dual-flank xenograft model employing λ secreting cells, by qPCR there was a reduction in λ LC mRNA with si[IGLCλCR] treatment in 13 of 16 mice (ALMC-1 11/114, ALMC-2 2/2). In these 14, the median λ LC expression was 66% of control (range, 17-97). In 6/13 the average reduction in λ LC expression was 59%. Of note, measurable levels of human λ LC were found in the blood of all mice at sacrifice. Conclusion With one pool of siRNA targeting the constant region of the κ or λ LC we can significantly reduce production and secretion of LC by clonal human plasma cells, including patient cells, and also reduce the expression of LC in xenograft plasmacytomas in vivo. Two methods of siRNA delivery have been employed in this work thus far, SLO and in vivo electroporation, neither of which require endosomal escape. The specificity of the siRNA pools for plasma cell LC genes and the possible receptivity of plasma cells to RNAi are important positive aspects of this work. Further pre-clinical development of Ig LC CR RNAi employing lipid-based nanoparticle platforms is warranted in order to optimize cell-specific delivery, delivery efficiency and siRNA targeting. Disclosures No relevant conflicts of interest to declare.

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Flow Cytometry Assessment of In Vitro Generated CD138+Human Plasma Cells

BioMed Research International ◽

10.1155/2014/536482 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 3

Author(s):

Rayelle Itoua Maïga ◽

Jennifer Lemieux ◽

Annie Roy ◽

Carl Simard ◽

Sonia Néron

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Human Plasma ◽

B Lymphocytes ◽

Cell Lines ◽

Mononuclear Cells ◽

Plasma Cells ◽

Peripheral Blood Mononuclear

The in vitro CD40-CD154 interaction promotes human B lymphocytes differentiation into plasma cells. Currently, CD138 is the hallmark marker enabling the detection of human plasma cells, both in vitro and in vivo; its presence can be monitored by flow cytometry using a specific antibody. We have developed a culture system allowing for the differentiation of memory B lymphocytes. In order to detect the newly formed plasma cells, we have compared their staining using five anti-CD138 monoclonal antibodies (mAbs). As a reference, we also tested human cell lines, peripheral blood mononuclear cells, and bone marrow samples. The five anti-CD138 mAbs stained RPMI-8226 cells (>98%) with variable stain index (SI). The highest SI was obtained with B-A38 mAb while the lowest SI was obtained with DL-101 and 1D4 mAbs. However, the anti-CD138 mAbs were not showing equivalent CD138+cells frequencies within the generated plasma cells. B-A38, B-B4, and MI-15 were similar (15–25%) while DL-101 mAb stained a higher proportion of CD138-positive cells (38–42%). DL-101 and B-A38 mAbs stained similar populations in bone marrow samples but differed in their capacity to bind toCD138highandCD138locell lines. In conclusion, such cellular fluctuations suggest heterogeneity in human plasma cell populations and/or in CD138 molecules.

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A New Xenograft Model for Studying Multiple Myeloma and Drug Response

Blood ◽

10.1182/blood.v116.21.4073.4073 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4073-4073

Author(s):

Varda Deutsch ◽

Yona Farnoushi ◽

Michal Cipok ◽

Sigi Kay ◽

Elizabeth Naparstek ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Cell Lines ◽

Conflicts Of Interest ◽

Xenograft Model ◽

In Vivo Model ◽

Pcr Analysis ◽

Human Leukemia ◽

In Vivo Models

Abstract Abstract 4073 While new treatment options are available, multiple myeloma (MM) still remains an incurable malignancy of plasma cells with a grim prognosis. Practical in vivo models to study human MM may enable a better understanding of the biology of the disease, and better optimization of therapeutic strategies. The best current xenograft model, the immune-deficient NOD/SCID mice, recapitulates MM in vivo, however, the price is very costly and maintenance complex, with >1 month required to establish engraftment. Our goal was to develop a user friendly rapid alternative xenograft system for the preclinical assessment of MM growth and therapy. We recently described this new in-vivo system for studying human leukemia in the pre-immune turkey embryo 1,2. These embryos are inexpensive, require no maintenance, and are easily manipulated experimentally. Described here are the first attempts at application of this novel system to study MM and test therapies. Cell lines ARH-77 and CAG line and fresh patient cells (5 × 106/embryo) were injected IV into turkey egg chorioallantoic membrane veins on embryonic day E11. Engraftment of human cells in hematopoietic organs, bone marrow (BM) and liver was detected 7 days later (E18) by RTPCR, immunohistochemistry and flow cytometry and by circulating free light chain (6-25 mg/L) in the peripheral blood of 100% of the injected cell lines and 50% of patients myelomas. Treatment with Velcade (Bortezomib) or Revlimid IV on E13 (48 hours after MM cell injection), at drug levels that were precalibrated to be non-toxic to the developing embryonic BM, dramatically reduced engraftment, demonstrating the utility of this new model for testing drug activity in vivo. ARH-77 cells, detected by flow cytometry of the embryonic BM cells with anti-human CD19, CD38 and CD138, were inhibited from 8.5% engraftment to 0.72% after a single Velcade treatment, with an 18 fold decrease compared to untreated embryos in the ratio of human to avian cells in BM tissue. determined by Q-RT-PCR analysis of human alpha satellite and avian GAPDH DNA normalized per cell. Very similar results were obtained with Revlimid. The results presented suggest that with further work the turkey embryo model may provide an affordable, rapid and practical xenograft system in vivo for studying the biology of MM, for affordably testing MM therapies, as well for developing a new method for individualized patient screening for response or resistance to particular therapeutic agents. 1. Taizi M, Deutsch VR, Leitner A, Ohana A, Goldstein RS. A novel and rapid in vivo system for testing therapeutics on human leukemias. Exp Hematol. 2006;34:1698-1708. 2. Grinberg I, Reis A, Ohana A, et al. Engraftment of human blood malignancies to the turkey embryo: a robust new in vivo model. Leuk Res. 2009;33:1417-1426. Disclosures: No relevant conflicts of interest to declare.

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Targeting CD20 and CD22 with Rituximab in Combination with CMC-544 Results in Improved Anti-Tumor Activity Against Non-Hodgkin’s Lymphoma (NHL) Pre-Clinical Models.

Blood ◽

10.1182/blood.v106.11.1473.1473 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1473-1473 ◽

Cited By ~ 3

Author(s):

Francisco J. Hernandez-Ilizaliturri ◽

Suresh Devineni ◽

Sarika Arora ◽

Joy Knight ◽

Myron S. Czuczman

Keyword(s):

Cell Lines ◽

Median Survival ◽

Flow Cytometric Analysis ◽

Scid Mice ◽

In Vivo Studies ◽

Parp Cleavage ◽

Rank Test ◽

Anti Tumor Activity

Abstract CMC-544 is an IgG4 CD22-humanized targeted immunoconjugate of calicheamicin. Upon CD22 biding, CMC-544 internalizes inducing cell apoptosis in vitro/in vivo against NHL with minimal modulation of the immune system. In addition to direct cell-death, rituximab, anti-CD20 monoclonal antibody (mAb), can induce complement-dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC). Combination of mAbs directed against unique tumor-associated targets can potentially enhance anti-tumor activity. Objectives: To study the effects of targeting CD20 and CD22 using rituximab in combination with CMC-544. Material and Methods: For in vitro studies, a panel of NHL cell lines [Raji, SUDHL-4, SU-DHL-10, Ramos, and two rituximab resistant cell lines (RRCL), [2R and 4RH] were exposed to CMC-544 (0.4pg/ml to 0.4μg/ml) +/− Rituximab (10μg/ml) or appropriate controls for 24 or 48 hrs. DNA synthesis was quantified by [H3]Thymidine incorporation and apoptosis was detected by multiparameter flow cytometric analysis and Poly(ADP-ribose) polymerase (PARP) cleavage. For ADCC/CMC studies, 51Cr-labeled NHL cells were exposed to CMC-544 (0.4μg/ml) prior to treatment with rituximab (10mg/ml) and peripheral blood mononuclear cells (Effector: Target ratio 40:1) or human serum, respectively. 51Cr-release was measured and the percentage of lysis was calculated. Statistical analysis of results was performed using the Chi-square test. For in vivo studies, 6–8-week old SCID mice were inoculated via tail vein injection (iv) with Raji cells (1 × 106 on day 0). Animals were assigned to receive 8 doses of either saline, CMC-544 (40μg/kg/dose), rituximab (10mg/kg/dose iv), or rituximab alternating with CMC-544. Antibodies were administered iv every other day starting on day +5. Survival analysis was performed by Kaplan-Meier curves and p values calculated using log rank test. Results: Exposure of various NHL cells to CMC-544 resulted in a dose-dependent significant decrease in cell proliferation and apoptosis. Rituximab-associated CMC or ADCC was not further enhanced by CMC-544 in vitro. In vivo studies demonstrated that the mean survival for animals treated with CMC-544 increased when compared to control mice [64 days (95% C.I. 48–80; 22 days (95% C.I. 21–23), respectively, P=0.001]. To a lesser degree rituximab monotherapy resulted in significant anti-tumor effects and the median survival reached 44 days (95% C.I. 31–56). Notably, animals treated with rituximab in combination with CMC-544 had the longest median survival (not reached at +90 days, log rank P=0.007). Conclusions: Our data suggest targeting CD20 and CD22 by rituximab and CMC-544 resulted in a longer survival in lymphoma-bearing SCID mice. Clinical trials with this combination will be needed to confirm these results for the treatment of lymphoma patients

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Spontaneous Development of Plasmacytoid Tumors in Mice with Defective Fas–Fas Ligand Interactions

Journal of Experimental Medicine ◽

10.1084/jem.187.11.1825 ◽

1998 ◽

Vol 187 (11) ◽

pp. 1825-1838 ◽

Cited By ~ 124

Author(s):

Wendy F. Davidson ◽

Thomas Giese ◽

Torgny N. Fredrickson

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Cell Lines ◽

Plasma Cells ◽

Significant Proportion ◽

T Cell Subsets ◽

Cell Populations ◽

B Cell Malignancies

B cell malignancies arise with increased frequency in aging individuals and in patients with genetic or acquired immunodeficiency (e.g., AIDS) or autoimmune diseases. The mechanisms of lymphomagenesis in these individuals are poorly understood. In this report we investigated the possibility that mutations at the Fas (lpr) and Fasl (gld) loci, which prevent Fas-mediated apoptosis and cause an early onset benign lymphoid hyperplasia and autoimmunity, also predispose mice to malignant lymphomas later in life. Up to 6 mo of age, hyperplasia in lpr and gld mice results from the predominant accumulation of polyclonal T cell subsets and smaller numbers of polyclonal B cells and plasma cells. Here, we examined C3H-lpr, C3H-gld, and BALB-gld mice 6–15 mo of age for the emergence of clonal T and B cell populations and found that a significant proportion of aging mice exclusively developed B cell malignancies with many of the hallmarks of immunodeficiency-associated B lymphomas. By 1 yr of age, ∼60% of BALB-gld and 30% of C3H-gld mice had monoclonal B cell populations that grew and metastasized in scid recipients but in most cases were rejected by immunocompetent mice. The tumors developed in a milieu greatly enriched for plasma cells, CD23− B cells and immunodeficient memory T cells and variably depleted of B220+ DN T cells. Growth factor–independent cell lines were established from five of the tumors. The majority of the tumors were CD23− and IgH isotype switched and a high proportion was CD5+ and dull Mac-1+. Considering their Ig secretion and morphology in vivo, most tumors were classified as malignant plasmacytoid lymphomas. The delayed development of the gld tumors indicated that genetic defects in addition to the Fas/Fasl mutations were necessary for malignant transformation. Interestingly, none of the tumors showed changes in the genomic organization of c-Myc but many had one or more somatically-acquired MuLV proviral integrations that were transmitted in scid passages and cell lines. Therefore, insertional mutagenesis may be a mechanism for transformation in gld B cells. Our panel of in vivo passaged and in vitro adapted gld lymphomas will be a valuable tool for the future identification of genetic abnormalities associated with B cell transformation in aging and autoimmune mice.

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No cell is an island: circulating T cell:monocyte complexes are markers of immune perturbations

10.1101/411553 ◽

2018 ◽

Author(s):

Julie G. Burel ◽

Mikhail Pomaznoy ◽

Cecilia S. Lindestam Arlehamn ◽

Daniela Weiskopf ◽

Ricardo da Silva Antunes ◽

...

Keyword(s):

Flow Cytometry ◽

T Cell ◽

Immune Responses ◽

Human Blood ◽

Significant Proportion ◽

Complex Frequency ◽

The Common ◽

First Time ◽

High Resolution Microscopy

AbstractOur results highlight for the first time that a significant proportion of cell doublets in flow cytometry, previously believed to be the result of technical artefacts and thus ignored in data acquisition and analysis, are the result of true biological interaction between immune cells. In particular, we show that cell:cell doublets pairing a T cell and a monocyte can be directly isolated from human blood, and high resolution microscopy shows polarized distribution of LFA1/ICAM1 in many doublets, suggesting in vivo formation. Intriguingly, T cell:monocyte complex frequency and phenotype fluctuate with the onset of immune perturbations such as infection or immunization, reflecting expected polarization of immune responses. Overall these data suggest that cell doublets reflecting T cell-monocyte in vivo immune interactions can be detected in human blood and that the common approach in flow cytometry to avoid studying cell:cell complexes should be revisited.

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PRMT5 Inhibition Drives Therapeutic Vulnerability to BCL-2 Inhibition with Venetoclax and Provides Rationale for Combination Therapy in Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2019-130797 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 302-302 ◽

Cited By ~ 1

Author(s):

Fiona Brown ◽

Yang Zhang ◽

Claire Hinterschied ◽

Alexander Prouty ◽

Shelby Sloan ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Combination Treatment ◽

Cell Lymphoma ◽

Targeted Agents ◽

Mantle Cell ◽

Anti Tumor Activity

Mantle cell lymphoma (MCL) is an incurable B cell malignancy, defined by the t(11;14) translocation and comprises 3-6% of non-Hodgkin lymphomas diagnosed annually. MCL is associated with a poor prognosis due to emergence of resistance to immuno-chemotherapy and targeted agents. Due to the late median age of diagnosis, aggressive chemotherapy and stem cell transplantation are often not realistic options. The average overall survival of patients with MCL is 5 years and for the majority of patients who progress on targeted agents like ibrutinib, survival remains at a dismal 3-8 months. There is a major unmet need to identify new therapeutic approaches that are well tolerated by elderly patients to improve treatment outcomes and quality of life. Our group has identified the type II protein arginine methyltransferase enzyme, PRMT5, to be dysregulated in MCL and to promote growth and survival by supporting the cell cycle, PRC2 activity, and signaling via the BCR and PI3K/AKT pathways. We have developed first-in-class selective inhibitors of PRMT5 and, in collaboration with Prelude Therapeutics, we have demonstrated that novel SAM-competitive PRMT5 inhibitors provide potent anti-tumor activity in aggressive preclinical models of human MCL. Selective inhibition of PRMT5 in these models and MCL cell lines leads to disruption of constitutive PI3K/AKT signaling, dephosphorylation and nuclear translocation of FOXO1, and enhanced recruitment of this tumor suppressor protein to chromatin. We identified 136 newly emerged FOXO1-bound genomic loci following 48 hours of PRMT5 inhibition in the CCMCL1 MCL line by performing chromatin immunoprecipitation-seq analysis. These genes were markedly upregulated in CCMCL1 cells treated with the PRMT5 inhibitor PRT382 as determined by RNA-seq analysis. Among those genes, we identified and confirmed FOXO1 recruitment to the promoter of BAX, a pro-apoptotic member of the BCL2 family of proteins. Treatment of MCL cell lines (Granta-519, CCMCL1, Z-138, and SEFA) with the selective PRMT5 inhibitor PRT382 (10, 100nM) led to upregulation of BAX protein levels and induction of programmed cell death as measured by annexin V/PI staining and flow cytometry. We hypothesized that induction of BAX would trigger a therapeutic vulnerability to the BCL2 inhibitor venetoclax, and that combination PRMT5/BCL2 inhibitor therapy would drive synergistic cell death in MCL. Single agent and combination treatment with venetoclax and PRT382 was performed in eight MCL lines including a new cell line generated from our ibrutinib-refractory PDX model (SEFA) and IC50 and synergy scores were calculated. The Z-138 line was most sensitive to venetoclax (IC50<10nM) while CCMCL-1, SP53, JeKo-1, and Granta-519 demonstrated relative resistance (IC50>1uM). All lines reached an IC50 <1uM when co-treated with PRT382, with IC50 values ranging from 20 - 500nM. Combination treatments showed high levels of synergy (scores > 20) in 4 lines and moderate synergy (scores 10-20) in 2 lines. The two lines with the highest levels of synergy, Z-138 and SEFA, express high levels of BCL-2 and are Ibrutinib resistant. Overall there was a strong positive correlation between BCL2 expression and synergy score (r=0.707), and no correlation between PRMT5 expression and synergy score (r=0.084). In vivo evaluation in two preclinical MCL models (Granta-519 NSG mouse flank and an ibrutinib-resistant MCL PDX) showed therapeutic synergy with combination venetoclax/PRT382 treatment. In both models, mice were treated with sub-therapeutic doses of venetoclax and/or PRT543 (Granta) or PRT382 (IR-MCL PDX) and tumor burden assessed weekly via flank mass measurement (Granta) or flow cytometry (IR-MCL-PDX). Combination treatment with well-tolerated doses of venetoclax and PRMT5 inhibitors in both MCL in vivo models showed synergistic anti-tumor activity without evidence of toxicity. This preclinical data provides mechanistic rationale while demonstrating therapeutic synergy and lack of toxicity in this preclinical study and justifies further consideration of this combination strategy targeting PRMT5 and BCL2 in MCL in the clinical setting. PRT543, a selective PRMT5 inhibitor, has been advanced into clinical studies for the treatment of patients with solid tumors and hematologic malignancies, including MCL (NCT03886831). Disclosures Zhang: Prelude Therapeutics: Employment. Vaddi:Prelude Therapeutics: Employment. Scherle:Prelude Therapeutics: Employment. Baiocchi:Prelude: Consultancy.

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Human t(1;19)(q23;p13) pre-B acute lymphoblastic leukemia in mice with severe combined immunodeficiency

Blood ◽

10.1182/blood.v81.11.3052.bloodjournal81113052 ◽

1993 ◽

Vol 81 (11) ◽

pp. 3052-3062 ◽

Cited By ~ 1

Author(s):

FM Uckun ◽

JR Downing ◽

R Gunther ◽

LM Chelstrom ◽

D Finnegan ◽

...

Keyword(s):

Flow Cytometry ◽

Acute Lymphoblastic Leukemia ◽

Poor Prognosis ◽

Scid Mouse ◽

Lymphoblastic Leukemia ◽

Fusion Transcript ◽

Scid Mice ◽

Newly Diagnosed ◽

In Vivo Growth

Severe combined immunodeficient (SCID) mice were injected intravenously with 5 x 10(6) primary bone marrow (BM) blasts from newly diagnosed patients with E2A-PBX1 fusion transcript positive t(1;19)(q23;p13) pre- B acute lymphoblastic leukemia (ALL). A marked variation existed in the pattern and extent of leukemic cell engraftment in SCID mice challenged with t(1;19) pre-B ALL blasts. Blasts from some patients caused disseminated leukemia that was detected by histopathology and/or flow cytometry, whereas blasts from other patients produced occult leukemia that was only detected by flow cytometry and/or polymerase-chain reaction. Notably, the ability of primary t(1;19) pre-B ALL blasts to cause disseminated leukemia in SCID mice was associated with poor prognosis. Six of six patients whose blasts caused disseminated leukemia in SCID mice relapsed at a median of 7.8 months (range: 5.7 to 25.2 months). In contrast, the remaining four patients whose blasts did not engraft or only partially engrafted remain in complete remission at 28 to 47 months. A new E2A-PBX-1 fusion transcript positive t(1;19) pre- B ALL cell line (designated LC1;19) with the composite immunophenotype CD7-CD10+CD19+CD45-HLA-DR+C mu+ was established by expanding BM blasts from a SCID mouse, which died of human t(1;19) ALL at 7 weeks after inoculation of primary leukemic blasts from a t(1;19) ALL patient. This cell line caused disseminated and invariably fatal leukemia when greater than 10(4) cells were injected intravenously into SCID mice. Total body irradiation followed by syngeneic BM transplantation (BMT) showed limited efficacy against LC1;19 leukemia in SCID mice. To our knowledge, this study is the first to (1) examine the in vivo growth of primary t(1;19) pre-B ALL blasts in SCID mice and (2) show that leukemic blasts from a majority of newly diagnosed t(1;19) pre-B ALL patients cause disseminated human leukemia in SCID mice. Our results indicate that t(1;19) pre-B ALL is biologically heterogeneous with regard to its in vivo growth pattern in SCID mice, a feature that may be predictive of prognosis. The described LC1;19 SCID mouse model may prove particularly useful for designing more effective treatment strategies against poor-prognosis t(1;19) ALL.

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Therapeutic Targeting of CCR1 to Prevent Dissemination of Multiple Myeloma Plasma Cells

Blood ◽

10.1182/blood-2019-121974 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3099-3099

Author(s):

Mara N Zeissig ◽

Duncan R Hewett ◽

Krzysztof M Mrozik ◽

Vasilios Panagopoulos ◽

Monika Engelhardt ◽

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Keyword(s):

Flow Cytometry ◽

Disease Progression ◽

Mononuclear Cells ◽

Plasma Cells ◽

Primary Tumour ◽

Nsg Mice ◽

Rpmi 8226 ◽

Retention Signal

Introduction:Multiple myeloma (MM) disease progression is dependent on the ability of the MM plasma cells (PC) to leave the bone marrow (BM), re-enter the peripheral blood (PB) and disseminate to other BM sites. Previous studies show that expression of CXCL12 by BM stromal cells is crucial for MM PC retention within the BM. However, the mechanisms which overcome this retention signal enabling MM PC egress and dissemination via the PB are poorly understood. Previous studies in haematopoietic progenitor cells have demonstrated that CCL3 overcomes the CXCL12 retention signal to drive mobilisation to the PB (Lord et al. Blood 1995). Here, we examined the role of the CCL3 chemokine receptor CCR1 in driving MM PC dissemination. Methods and results: Initially, we assessed the expression of CCR1 protein on CD138+CD38++CD45loCD19- PC from 28 MM, 8 MGUS and 2 SMM patients by flow cytometry. Results show CCR1 expression is significantly increased in newly diagnosed MM compared with premalignant MGUS and SMM patients (p=0.03; CCR1 MFI mean±SEM, MGUS: 53.0±33.6; SMM: 37.6±8.9 MM: 250.9±71.6). Furthermore, CCR1 expression on PB MM PC positively correlated with PB MM PC numbers (p=0.03; n=11 patients). To identify mechanistically how CCR1 may promote dissemination, the effect of CCL3 on the response to CXCL12 in human myeloma cell lines (HMCL) was assessed in vitro. The migration of RPMI-8226 and OPM2 cells was induced by CCL3 or CXCL12 chemoattractant in a transwell assay. Notably, pre-treatment of RPMI-8226 or OPM2 with CCL3 abrogated migration towards CXCL12 and blocked F-actin remodelling in response to CXCL12 in vitro. These findings suggest that CCL3 can desensitise cells to exogenous CXCL12, providing a potential mechanism facilitating loss of the CXCL12 retention signal. To confirm whether CCR1 is required for driving MM PC dissemination, homozygous CCR1 knockout (KO) cells were generated using a lentiviral CRISPR/Cas9 system in OPM2 cells. CCR1-KO OPM2 cells were confirmed to have no detectable CCR1 expression by flow cytometry and could no longer migrate towards CCL3 in vitro. Empty vector (EV) or CCR1-KO OPM2 MM PC were injected into the tibia of immune-compromised NOD-scidgamma (NSG) mice. After 4 weeks, primary tumour within the injected tibia and disseminated tumour in the PB and the contralateral tibia and femur was assessed by flow cytometry. We found that mice bearing CCR1-KO cells have a 45.5% decrease in primary tumour growth (p=0.008; % GFP+ of total mononuclear cells, EV: 77.2±17.2; CCR1-KO: 42.1±24.4), a 97.8% reduction in PB MM PC (p<0.0001; EV: 1.39±0.7; CCR1-KO: 0.03±0.046) anda 99.9% reduction in BM tumour dissemination (p<0.0001; EV: 49.5±17; CCR1-KO: 0.019±0.013), compared with controls. In a supportive study, CCR1 was expressed in the murine MM cell line 5TGM1 using lentiviral transduction. 5TGM1-CCR1 cells were confirmed to express CCR1 by qPCR and were able to migrate towards CCL3 in vitro. 5TGM1-CCR1 or EV cells were injected into the tibiae of C57BL/KaLwRij mice and allowed to initiate systemic MM disease for 3.5 weeks. Importantly, while 55% of control mice exhibited disseminated tumours, this increased to 92% with CCR1 expression (p<0.0001; n=12/group). These data suggest that CCR1 expression on MM PC may play an important role in MM PC dissemination. To determine whether therapeutic inhibition of CCR1 prevents dissemination, the effect of a small molecule CCR1 inhibitor, CCR1i, was assessed in vivo. OPM2 EV or RPMI-8226 cells were injected into the tibia of NSG mice and, after 3 days, mice were treated with CCR1i (15mg/kg) or vehicle twice daily by oral gavage for 25 days. OPM2-inoculated CCR1i-treated mice had 66.1% lower PB MM PC (p<0.0001; % GFP+ of total mononuclear cells, vehicle: 23.9±7.2; CCR1i: 8.1±3.8) and a 22.1% reduction in BM dissemination (p=0.0002; vehicle: 78.1±4.8;CCR1i: 60.8±7.1) compared with controls. Similarly, CCR1i treatment reduced BM dissemination by 59.6% in RPMI-8226 bearing mice (p<0.0001; % GFP+ of total mononuclear cells, vehicle: 0.86±0.15; CCR1i: 0.26±0.05). This suggests that CCR1 inhibition can slow tumour dissemination in vivo. Conclusion:This study identified CCR1 as a novel driver of MM PC dissemination in vivo, at least in part by overcoming the CXCL12 retention signal. Importantly, this study demonstrated for the first time that targeting CCR1 can be a viable therapeutic strategy to limit dissemination and potentially slow disease progression. Disclosures Croucher: Trovagene: Employment.

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