scholarly journals Anti-CD37 Alpha-Amanitin Conjugated Antibodies As Therapeutic Weapons for Richter's Syndrome

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 791-791
Author(s):  
Tiziana Vaisitti ◽  
Nicoletta Vitale ◽  
Andrea Iannello ◽  
Lorenzo Brandimarte ◽  
Matilde Micillo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Rna Polymerase ◽  
B Cell ◽  
Rna Polymerase Ii ◽  
Rna Sequencing ◽  
Research Funding ◽  
Single Administration ◽  
Alpha Amanitin ◽  
Pdx Models ◽  
Richter’S Syndrome

Abstract The leukocyte surface antigen CD37 (TSPAN26), a member of the tetraspanin superfamily, is widely expressed on most malignant B cells, making it an actionable target for treatment of patients with chronic lymphocytic leukemia (CLL) and other B-cell non-Hodgkin lymphoma (NHL) indications. Accordingly, αCD37 antibodies have shown promising results in phase 1/2 clinical trials for CLL and NHL. Richter's syndrome (RS) is the transformation of CLL into an aggressive and rapidly fatal lymphoma, typically a diffuse large B cell lymphoma (DLBCL). RS is a challenging disease since very few effective treatment options exist for these patients and the available regimens, mainly based on R-CHOP scheme, show limited efficacy. We recently established patient-derived xenograft (PDX) models from RS patients and have shown that they can be used to test the efficacy of novel drugs and drug combinations 1,2. All available RS-PDX models were characterized by high-levels of CD37 expression, when assessed by RNA sequencing, reverse-transcriptase-polymerase chain reaction (RT-PCR), flow cytometry (FACS), western blot (WB) and immunohistochemistry (IHC). More precisely, two models (RS1316 and IP867/17) showed slightly higher CD37 levels compared to the others (RS9737 and RS1050). These models were used to test three different αCD37-ATACs®, ADCs which comprise amanitin-derivatives as payload. Amanitin (the main poison in the green deathcap mushroom) belongs to the well-known amatoxin family. Amanitin is taken up by OATP1B3 transporter, solely expressed on hepatocytes. Upon mushroom intoxication, it can lead to severe liver toxicity by inhibiting the RNA polymerase II. Upon conjugation to target-specific antibodies, the maximal tolerated dose is significantly increased by reducing the non-specific liver uptake. By binding to its antigen, ATACs deliver amanitin only into target-positive cancer cells while target negative cells show no off-target toxicity. Consistent with CD37 expression on the cell surface, ex-vivo treatment of freshly purified cells from RS-PDX tumor masses to αCD37-ATACs® resulted in increased apoptosis after 72 hours of treatment, with only minor differences among the 3 ATACs® and the models used. Since alpha-amanitin is a deadly toxin known to target human RNA polymerase II and, at high doses, also RNA polymerase III, we checked messenger RNA levels in basal conditions and after CD37-ATAC® treatment by looking at different housekeeping genes, and confirmed a reduction in global mRNA levels. αCD37-ATAC® efficacy was then assessed in vivo in systemic RS-PDX models where RS cells are intra-venously (i.v.) injected in the tail vein and cells distribute to different tissues (blood, spleen and bone marrow), resembling the human disease. Cells from RS1316, RS1050 and RS9737 models were injected into the tail vein and left to engraft 14 days, before mice were randomly assigned to vehicle or ATAC® groups. A single i.v. treatment for each αCD37-ATAC® was administered, testing two different doses for each compound, and mice were then monitored for survival. Overall, the single administration of all three ATACs® caused highly significant disease regression. In the RS1316 model, independently of the dose or tested ATAC®s, all treated mice, except one, were alive and disease-free until the end of the experiment, 140 days post cells injection, while survival of vehicle-treated mice was 65 days. FACS analysis to trace neoplastic cells in parenchymatous organs and bone marrow confirmed the absence of neoplastic cells. In the other 2 models, RS9737 and RS1050, even though ATAC® treatment did not result in complete disease eradication, a single administration of αCD37-ATAC® resulted in a dramatically increased survival (approximately 35-60 days, depending on the model and ATAC® used). Finally, CD37 expression was confirmed by RNA sequencing on a cohort of 15 primary RS samples, even though with variable levels. Compared to CLL cells, RS samples showed CD37 expression levels comparable to those reported for DLBCL cells. Overall, these data indicate CD37 as a potential target to treat RS patients with selective targeting αCD37-ATACs®. ATACs® is a registered trade mark of Heidelberg Pharma Research GmbH, Germany References  Vaisitti T et al. Blood. 2021;137(24):3365-3377. Iannello A, et al. Blood. 2021;137(24):3378-3389. Disclosures Orlik: Heidelberg Pharma: Current Employment. Kulke: Heidelberg Pharma: Current Employment. Pahl: Heidelberg Pharma: Current Employment. Deaglio: Heidelberg Pharma: Research Funding; Astra Zeneca: Research Funding.

scholarly journals Evidence of a Synergistic Cross-Talk between the B Cell Receptor (BCR) and Nicotinamide Phosphoribosyl Transferase (NAMPT) in Richter's Syndrome Patient-Derived Xenograft Models: Therapeutic Implications

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 250-250
Author(s):  
Vincenzo Gianluca Messana ◽  
Nicoletta Vitale ◽  
Matteo Rovere ◽  
Lucia Renzullo ◽  
Francesca Arruga ◽  
...  
Keyword(s):  
B Cell ◽  
Disease Progression ◽  
Research Funding ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Metabolic Reprogramming ◽  
Rate Limiting ◽  
Pdx Models ◽  
Richter’S Syndrome

Abstract Background: A rare complication of Chronic Lymphocytic Leukemia (CLL), Richter's Syndrome (RS) represents the transformation of a pre-existing CLL into a Diffuse Large B-cell Lymphoma (DLBCL), generally associated with poor prognosis. Current therapeutic approaches are limited and do not significantly reduce disease progression. For these reasons there is intense investigation to identify potentially druggable molecular circuits, opening the way to innovative combination therapies. Among the several oncogenic signalling pathways that may contribute to disease progression, the B Cell Receptor (BCR) is a main driver and an actionable target. We previously showed that BCR ligation in CLL cells increases expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the salvage NAD pathway starting from nicotinamide, a finding in line with the notion of oncogenic-driven metabolic reprogramming. In this context, increased NAMPT expression leading to heightened NAD+ levels could sustain proliferation through the modulation of the activity of several intracellular enzymes, including sirtuins (NAD-dependent deacetylases). Aim: This work explores the connections between BCR signalling and the NAMPT/NAD/sirtuin axis in RS cells, asking the question of whether BCR and NAMPT can be simultaneously targeted. Methods: RS-PDX cells were freshly purified from tumor masses grown in mice and immediately used for short ex-vivo experiments. For in vivo experiments, NSG mice were injected subcutaneously with RS-PDX cells, which were left to engraft until a palpable mass was evident. Mice were then randomized to receive duvelisib, OT82 or a combination of the two for two consecutive weeks. Control mice were similarly treated with vehicle only. Mice were under a niacin-free diet. Results: By using our 4 RS patient-derived xenografts (PDX) models, we invariably observed high levels of NAMPT expression. High levels of NAMPT expression were also observed in 15 primary RS lymph node biopsies analyzed by RNA sequencing. BCR engagement through αIgM polyclonal antibodies significantly up-regulated NAMPT expression, as determined by qRT-PCR and protein analysis, with a concomitant increase in intracellular NAD+ levels. We then asked whether RS cells are sensitive to NAMPT inhibition, alone or in combination with drugs that target the BCR pathway. As most RS patients would likely have been treated during the preceding CLL phase with a BTK inhibitor, possibly developing resistance, we turned to PI3K inhibitors, which are less commonly used for CLL therapy. As NAMPT inhibitors (NAMPTi) we used both FK866 and OT-82, which are validated small molecules. Results indicated that the combination of the dual PI3K-δ/γ inhibitor duvelisib, with either FK866 or OT-82 induces dramatic apoptosis in all 4 models tested, as confirmed by annexinV/PI staining, by caspase 3 activation and by a significant drop in ATP and NAD+ levels. Importantly, two RS-PDX models (RS9737 and RS1316) were fully resistant to NAMPTi used alone, likely due to high levels of nicotinate phosphoribosyltransferase (NAPRT), which is the rate-limiting enzyme in the NAD salvage pathway that starts from nicotinic acid. However, addition of duvelisib, which was mildly effective when used alone, was followed by marked apoptosis even in these two models. Molecular dissection of the pathway showed that the combination of duvelisib and NAMPTi was followed by complete inhibition of the PI3K pathway, which was only partially blocked by duvelisib alone, even at high doses. The connection between NAMPT and PI3K is represented by cytoplasmic sirtuins, particularly SIRT2, which activate AKT through de-acetylation. Immunoprecipitation and two-dimensional gel electrophoresis showed that in the presence of NAMPTi, the amount of acetylated, i.e., inactive, AKT increased considerably. Consistently, treatment of RS-PDX mice with a combination of duvelisib and OT-82 was followed by significantly higher responses and longer animal survival. Conclusions: These results highlight a crosstalk between BCR signalling and NAMPT/sirtuin axis in RS models, showing the increased efficacy of the dual targeting (i.e., PI3K-δ/γ and NAMPT), and supporting this novel and promising therapeutic strategy for the treatment of RS patients. Disclosures Deaglio: Heidelberg Pharma: Research Funding; Astra Zeneca: Research Funding.

scholarly journals Statistical inference of the rate of RNA polymerase II elongation by total RNA sequencing

2018 ◽  
Vol 35 (11) ◽  
pp. 1877-1884
Author(s):  
Yumi Kawamura ◽  
Shinsuke Koyama ◽  
Ryo Yoshida
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Statistical Inference ◽  
Rna Sequencing ◽  
Total Rna

scholarly journals Phase II Trial of the Combination of Ixazomib, Lenalidomide, and Dexamethasone in High-Risk Smoldering Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 804-804 ◽  
Author(s):  
Mark Bustoros ◽  
Chia-jen Liu ◽  
Kaitlen Reyes ◽  
Kalvis Hornburg ◽  
Kathleen Guimond ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
High Risk ◽  
Disease Progression ◽  
Board Of Directors ◽  
Rna Sequencing ◽  
Research Funding ◽  
Response Rate ◽  
Advisory Committees ◽  
Equity Ownership

Abstract Background. This study aimed to determine the progression-free survival and response rate using early therapeutic intervention in patients with high-risk smoldering multiple myeloma (SMM) using the combination of ixazomib, lenalidomide, and dexamethasone. Methods. Patients enrolled on study met eligibility for high-risk SMM based on the newly defined criteria proposed by Rajkumar et al., Blood 2014. The treatment plan was designed to be administered on an outpatient basis where patients receive 9 cycles of induction therapy of ixazomib (4mg) at days 1, 8, and 15, in combination with lenalidomide (25mg) at days 1-21 and Dexamethasone at days 1, 8, 15, and 22. This induction phase is followed by ixazomib (4mg) and lenalidomide (15mg) maintenance for another 15 cycles. A treatment cycle is defined as 28 consecutive days, and therapy is administered for a total of 24 cycles total. Bone marrow samples from all patients were obtained before starting therapy for baseline assessment, whole exome sequencing (WES), and RNA sequencing of plasma and bone marrow microenvironment cells. Moreover, blood samples were obtained at screening and before each cycle to isolate cell-free DNA (cfDNA) and circulating tumor cells (CTCs). Stem cell collection is planned for all eligible patients. Results. In total, 26 of the planned 56 patients were enrolled in this study from February 2017 to April 2018. The median age of the patients enrolled was 63 years (range, 41 to 73) with 12 males (46.2%). Interphase fluorescence in situ hybridization (iFISH) was successful in 18 patients. High-risk cytogenetics (defined as the presence of t(4;14), 17p deletion, and 1q gain) were found in 11 patients (61.1%). The median number of cycles completed was 8 cycles (3-15). The most common toxicities were fatigue (69.6%), followed by rash (56.5%), and neutropenia (56.5%). The most common grade 3 adverse events were hypophosphatemia (13%), leukopenia (13%), and neutropenia (8.7%). One patient had grade 4 neutropenia during treatment. Additionally, grade 4 hyperglycemia occurred in another patient. As of this abstract date, the overall response rate (partial response or better) in participants who had at least 3 cycles of treatment was 89% (23/26), with 5 Complete Responses (CR, 19.2%), 9 very good partial responses (VGPR, 34.6%), 9 partial responses (34.6%), and 3 Minimal Responses (MR, 11.5%). None of the patients have shown progression to overt MM to date. Correlative studies including WES of plasma cells and single-cell RNA sequencing of the bone microenvironment cells are ongoing to identify the genomic and transcriptomic predictors for the differential response to therapy as well as for disease evolution. Furthermore, we are analyzing the cfDNA and CTCs of the patients at different time points to investigate their use in monitoring minimal residual disease and disease progression. Conclusion. The combination of ixazomib, lenalidomide, and dexamethasone is an effective and well-tolerated intervention in high-risk smoldering myeloma. The high response rate, convenient schedule with minimal toxicity observed to date are promising in this patient population at high risk of progression to symptomatic disease. Further studies and longer follow up for disease progression are warranted. Disclosures Bustoros: Dava Oncology: Honoraria. Munshi:OncoPep: Other: Board of director. Anderson:C4 Therapeutics: Equity Ownership; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Takeda Millennium: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncopep: Equity Ownership. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Ghobrial:Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; BMS: Consultancy.

scholarly journals A DNA-dependent RNA synthesis by wheat-germ RNA polymerase II insensitive to the fungal toxin α-amanitin

1992 ◽  
Vol 285 (1) ◽  
pp. 85-90 ◽  
Author(s):  
C Job ◽  
D Shire ◽  
V Sure ◽  
D Job
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Rna Synthesis ◽  
Biochemical Properties ◽  
Size Analysis ◽  
Reaction Products ◽  
High Concentration ◽  
Fungal Toxin ◽  
Alpha Amanitin

Wheat-germ RNA polymerase II is able to catalyse a DNA-dependent reaction of RNA synthesis in the presence of a high concentration (1 mg/ml) of the fungal toxin alpha-amanitin. This anomalous reaction is specifically directed by single-stranded or double-stranded homopolymer templates, such as poly(dC) or poly(dC).poly(dG), and occurs in the presence of either Mn2+ or Mg2+ as the bivalent metal cofactor. In contrast, the transcription of other synthetic templates, such as poly(dT), poly(dA).poly(dT) or poly[d(A-T)] is completely abolished in the presence of 1 microgram of alpha-amanitin/ml, in agreement with well-established biochemical properties of class II RNA polymerases. Size analysis of reaction products resulting from transcription of (dC)n templates of defined lengths suggests that polymerization of RNA chains proceeds through a slippage mechanism. The fact that alpha-amanitin does not impede this synthetic reaction implies that the amatoxin interferes with the translocation of wheat-germ RNA polymerase II along the DNA template.

scholarly journals Localization of an alpha-amanitin resistance mutation in the gene encoding the largest subunit of mouse RNA polymerase II.

1987 ◽  
Vol 7 (2) ◽  
pp. 586-594 ◽  
Author(s):  
M S Bartolomei ◽  
J L Corden
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Line ◽  
Resistance Mutation ◽  
Wild Type Mouse ◽  
Mutant Form ◽  
Wild Type ◽  
Gene Encoding ◽  
Base Pair Fragment ◽  
Alpha Amanitin

RNA polymerase II is inhibited by the mushroom toxin alpha-amanitin. A mouse BALB/c 3T3 cell line was selected for resistance to alpha-amanitin and characterized in detail. This cell line, designated A21, was heterozygous, possessing both amanitin-sensitive and -resistant forms of RNA polymerase II; the mutant form was 500 times more resistant to alpha-amanitin than the sensitive form. By using the wild-type mouse RNA polymerase II largest subunit (RPII215) gene (J.A. Ahearn, M.S. Bartolomei, M. L. West, and J. L. Corden, submitted for publication) as the probe, RPII215 genes were isolated from an A21 genomic DNA library. The mutant allele was identified by its ability to transfer amanitin resistance in a transfection assay. Genomic reconstructions between mutant and wild-type alleles localized the mutation to a 450-base-pair fragment that included parts of exons 14 and 15. This fragment was sequenced and compared with the wild-type sequence; a single AT-to-GC transition was detected at nucleotide 6819, corresponding to an asparagine-to-aspartate substitution at amino acid 793 of the predicted protein sequence. Knowledge of the position of the A21 mutation should facilitate the study of the mechanism of alpha-amanitin resistance. Furthermore, the A21 gene will be useful for studying the phenotype of site-directed mutations in the RPII215 gene.

Zinc deficiency and the Euglena gracilis chromatin: formation of an .alpha.-amanitin-resistant RNA polymerase II

Biochemistry ◽  
1985 ◽  
Vol 24 (10) ◽  
pp. 2576-2580 ◽  
Author(s):  
Kenneth H. Falchuk ◽  
Barbara Mazus ◽  
Elzbieta Ber ◽  
Leslie Ulpino-Lobb ◽  
Bert L. Vallee
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Zinc Deficiency ◽  
Euglena Gracilis ◽  
Alpha Amanitin
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