Synthetic lethality of LP-184, a next generation acylfulvene, in ex vivo PDX models with homologous recombination defects.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e15064-e15064
Author(s):  
Aditya Kulkarni ◽  
Diana Restifo ◽  
Igor A. Astsaturov ◽  
Umesh Kathad ◽  
Joseph McDermott ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Cell Line ◽  
Cell Lines ◽  
Synthetic Lethality ◽  
Ex Vivo ◽  
Clinical Success ◽  
Tumor Growth Inhibition ◽  
Cytotoxic Action ◽  
Cancer Type ◽  
Pdx Models

e15064 Background: The clinical success of PARP inhibitors (PARPi) in homologous recombination (HR) deficient (HRD+) solid tumors has broadened the scope of identifying additional agents and vulnerabilities in cancers with DNA repair deficiencies. However, with more than 40% of BRCA1/2-deficient patients failing to respond to PARPi or acquiring resistance with prolonged PARPi administration, newer agents are also needed. LP-184, an acylfulvene, is a prodrug activated by PTGR1. Threshold expression levels of PTGR1 are higher in several tumors, providing a window of specificity for its cytotoxic action. DNA damage inflicted by acylfulvene (AF) agents is reliant upon HR pathway genes including BRCA1 for correction and removal. We hypothesized that tumors with high PTGR1 expression and HR deficiency will therefore be uniquely targeted and demonstrate synthetic lethality when exposed to LP-184. Methods: We evaluated ex vivo antitumor activity of LP-184 in selected PDX models representing lung, pancreatic and prostate cancers with high PTGR1 and known HR defects. Dissociated tumor fragments were treated with LP-184 across a concentration range of 5 nM to 36 uM for 5 days. Cell viability was quantified by CellTiter Glo. LP-184 IC50s were compared with PARPi efficacy. We further confirmed the dependency of PTGR1 in HR deficient tumor cells by comparing LP-184 sensitivity in the BRCA2 deficient cell line CAPAN-1 and the ATM mutant cell line PANC03.27, with and without PTGR1 suppression following an engineered CRISPR knockout of PTGR1. We also analyzed TCGA data to estimate the percentage of tumors with elevated PTGR1 and co-occurring damaging mutations in a panel of 60 HR genes. Results: The mean LP-184 IC50 across 15 HRD+ PDX models tested was 288 nM (range 31 - 2900 nM). LP-184 turned out to be 6 - 340X more potent ex vivo than the PARPi Olaparib in these models. 9 of 15 models were associated with no clinical response to or initial response followed by progression on approved standard of care (SOC) agents. 6 of 15 models showed < 10% tumor growth inhibition in vivo with SOC treatment. Regardless of cancer type, models with high-impact, loss-of-function mutations in ATM, ATR and BRCA1 showed exquisite sensitivity to LP-184 (mean IC50 ̃ 60 nM). CRISPRi-mediated stable suppression of PTGR1 in the pancreatic cancer cell lines CAPAN-1 and PANC03.27 entirely abrogated LP-184 sensitivity relative to isogenic parental cell lines. 17.6% of lung adenocarcinomas (n = 517), 4.5% of pancreatic adenocarcinomas (n = 179) and 9.6% of prostate adenocarcinomas (n = 498) displayed elevated PTGR1 along with damaging HR related mutations, and are likely to be responsive to LP-184 based on analysis of TCGA data. Conclusions: LP-184 is broadly effective in HRD+ tumors that may be less responsive to SOC including PARPi and could be useful clinically in a subset of tumors with high PTGR1 and HR defects.

scholarly journals Infection of Cell Lines with Experimental and Natural Ovine Scrapie Agents

2009 ◽  
Vol 84 (5) ◽  
pp. 2444-2452 ◽  
Author(s):  
Michael H. Neale ◽  
Susan J. Mountjoy ◽  
Jane C. Edwards ◽  
Didier Vilette ◽  
Hubert Laude ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Ex Vivo ◽  
Transmissible Spongiform Encephalopathy ◽  
Attractive Alternative ◽  
Mouse Bioassay ◽  
Spongiform Encephalopathy ◽  
Parental Line ◽  
Biological Origin ◽  
Infectious Titer

ABSTRACT Mouse bioassay remains the gold standard for determining proof of infectivity, strain type, and infectious titer estimation in prion disease research. The development of an approach using ex vivo cell-based assays remains an attractive alternative, both in order to reduce the use of mice and to hasten results. The main limitation of a cell-based approach is the scarcity of cell lines permissive to infection with natural transmissible spongiform encephalopathy strains. This study combines two advances in this area, namely, the standard scrapie cell assay (SSCA) and the Rov9 and MovS6 cell lines, which both express the ovine PrP VRQ allele, to assess to what extent natural and experimental ovine scrapie can be detected ex vivo. Despite the Rov9 and MovS6 cell lines being of different biological origin, they were both permissive and resistant to infection with the same isolates of natural sheep scrapie as detected by SSCA. Rov9 subclones that are 20 times more sensitive than Rov9 to SSBP/1-like scrapie infection were isolated, but all the subclones maintained their resistance to isolates that failed to transmit to the parental line. The most sensitive subclone of the Rov9 cell line was used to estimate the infectious titer of a scrapie brain pool (RBP1) and proved to be more sensitive than the mouse bioassay using wild-type mice. Increasing the sensitivity of the Rov9 cell line to SSBP/1 infection did not correlate with broadening susceptibility, as the specificity of permissiveness and resistance to other scrapie isolates was maintained.

Partially or Fully BCR-ABL Independent Mechanisms of in Vitro Resistance to Ponatinib

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2481-2481 ◽  
Author(s):  
Qian Yu ◽  
Anna M Eiring ◽  
Matthew S. Zabriskie ◽  
Jamshid Khorashad ◽  
David J Anderson ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Synthetic Lethality ◽  
Immunoblot Analysis ◽  
Kinase Domain ◽  
Resistant Cell ◽  
Abl Kinase ◽  
Kinase Domain Mutation

Abstract Abstract 2481 Ponatinib (AP24534) is a pan-BCR-ABL inhibitor developed for treatment-refractory chronic myeloid leukemia (CML) and has significant activity in patients who fail second-line dasatinib and/or nilotinib tyrosine kinase inhibitor (TKI) therapy. A pivotal phase II trial (clinicaltrials.gov NCT01207440) is underway. BCR-ABL kinase domain mutation-mediated ponatinib resistance has been investigated in vitro (Cancer Cell 16, 2009, 401). Here, we developed ponatinib-resistant, BCR-ABL+ cell lines lacking a kinase domain mutation and investigated mechanisms of resistance to ponatinib and other TKIs. Methods: Four BCR-ABL+ CML cell lines (K562, AR230, BV173, and 32D(BCR-ABL)) were maintained in liquid culture containing ponatinib (0.1 nM) for 10 days. The ponatinib concentration was increased in small increments for a minimum of 90 days, yielding corresponding ponatinib-resistant cell lines. BCR-ABL kinase domain sequencing of sensitive and resistant cells confirmed BCR-ABL to be unmutated. Real-time qPCR was used to compare the expression of BCR-ABL in ponatinib-sensitive and -resistant cell lines. Immunoblot analysis (total and tyrosine-phosphorylated BCR-ABL) was used to the compare levels of BCR-ABL protein and to determine whether resistance to ponatinib corresponded with reduced (partially BCR-ABL-independent) or complete inhibition of BCR-ABL tyrosine phosphorylation (fully BCR-ABL-independent). Cell proliferation assays were performed on resistant and sensitive cell lines in the presence of ponatinib, nilotinib, and dasatinib. A small-molecule inhibitor screen composed of >90 cell-permeable inhibitors that collectively target the majority of the tyrosine kinome as well as other kinases (Blood 116, 2010, abstract 2754) is currently being applied to the 32D(BCR-ABL)R cell line in the presence of 24 nM ponatinib to assess synthetic lethality, with results analyzed using a companion drug sensitivity algorithm. As a second strategy to generate resistant lines, N-ethyl-N-nitrosourea (ENU) mutagenesis was done to investigate BCR-ABL kinase domain-mediated resistance in myeloid K562, AR230, BV173, and 32D(BCR-ABL) cells. After ENU exposure, cells were washed and cultured in 96-well plates with escalating ponatinib. Results: The four BCR-ABL+ cell lines initially grew in the presence of 0.1 nM but not 0.5 nM ponatinib. Upon gradual exposure to escalating ponatinib, each of the cell lines exhibited a degree of adaptation to growth in the presence of the inhibitor (range: 10 to 240-fold). Real-time qPCR showed a modest two-fold increase in BCR-ABL expression level in K562R, AR230R and BV173R cell lines relative to the respective parental lines. Based on immunoblot analysis, cell lines segregated into two categories of ponatinib resistance: partially (K562R and AR230R) or fully BCR-ABL-independent (BV173R and 32D(BCR-ABL)R). Cell proliferation assays showed that ponatinib resistant cell lines also exhibited resistance to nilotinib and dasatinib. The 32D(BCR-ABL)R cell line exhibited a level of ponatinib resistance comparable to that of the Ba/F3 BCR-ABLE255V cell line, which carries the most ponatinib-resistant BCR-ABL mutation. BCR-ABL tyrosine phosphorylation was efficiently blocked by low concentrations of ponatinib (<5 nM) in the 32D(BCR-ABL)R cell line, yet these cells remained viable in the presence of up to 24 nM ponatinib. The effects of providing a second kinase inhibitor along with ponatinib (24 nM) in order to probe for synthetic lethality are under study. Possible involvement of a second, moderately ponatinib-sensitive target is suggested by the sharp ponatinib maximum at 24 nM; even 26 nM ponatinib is toxic to 32D(BCR-ABL)R cells. Thus far, ENU mutagenesis screens in human CML cell lines failed to yield resistant clones and only a few were recovered from the murine 32D(BCR-ABL)R cell line (3/1440 wells; the only BCR-ABL mutant recovered was BCR-ABLL387F). Conclusions: The ponatinib resistant, BCR-ABL+ cell lines described here exhibit either a partially or fully BCR-ABL independent mechanism of resistance. The molecular details of both processes will be reported, with an emphasis on the striking level of resistance (240-fold over starting conditions) exhibited by the 32D(BCR-ABL)R cell line. Our in vitro results indicate that BCR-ABL independent mechanisms may contribute to ponatinib resistance in myeloid CML cells. Disclosures: No relevant conflicts of interest to declare.

Identification of Ex Vivo Myeloma Cells with the PNH Phenotype

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3994-3994
Author(s):  
David J Araten ◽  
Ken Csehak ◽  
Leah Zamechek ◽  
Jeesun Park ◽  
Cynthia Liu ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Ex Vivo ◽  
Transmembrane Proteins ◽  
Single Mutation ◽  
Myeloma Cells ◽  
Prior Therapy ◽  
Oncogenic Mutations ◽  
Mantle Cell ◽  
Inactivating Mutations

Abstract Abstract 3994 Myeloma cells can harbor ∼ 35 mutations, whereas according to Loeb's model, if > 2 oncogenic mutations are required for the development of a malignancy, then an elevation in the mutation rate would be required for these mutations to occur in the same cell. Indeed, there is evidence that hypermutability in myeloma may be a result of abnormal homologous recombination or activation induced cytidine deaminase. An alternative model is that successive rounds of clonal selection in an expanding pre-malignant population could result in an accumulation of oncogenic mutations. Using our method for the detection of rare cells that have an acquired somatic mutation of the PIG-A gene, we have previously demonstrated hypermutability in some but not all lymphoma and myeloma cell lines, and we have recently shown that samples of ex vivo blasts from ∼ 50% of patients with ALL demonstrate hypermutability. Here we have hypothesized that genomic instability could be identified in myeloma. PIG-A encodes an enzyme that is necessary for the biosynthesis of glycosylphosphatidylinositol (GPI) and is particularly promising as a sentinel gene for spontaneous mutations because it is X-linked, and thus a single mutation can produce the mutant phenotype. PIG-A is mutated in Paroxysmal Nocturnal Hemoglobinuria (PNH), and it is known from this condition that a broad spectrum of inactivating mutations can produce the PNH phenotype, which is a loss of all GPI-linked surface proteins. PIG-A mutations do not affect transmembrane proteins and are growth-neutral under almost all circumstances. The PNH phenotype can be readily detected by flow cytometry, using antibodies specific for GPI-linked proteins (e.g., CD48, CD55, and CD59), as well the FLAER reagent, which binds directly to GPI. In order to quantitate the frequency of myeloma cells with the PNH phenotype, we analyzed thawed ficolled samples from patients with a heavy burden of myeloma cells in the marrow. Cells were stained sequentially with FLAER-Alexa 488, then with a mixture of murine anti-CD48, anti-CD55, and anti-CD59 antibodies, followed by FITC-conjugated rabbit anti-mouse immunoglobulin, followed by a PE-conjugated antibody specific for a myeloma-specific transmembrane protein–either CD38 or CD138. Live myeloma cells were identified by forward/side scatter and propidium iodide exclusion and expression of CD38 or CD138. Using this approach, in previous studies we have seen that cells with the PNH phenotype have a low FITC/FLAER fluorescence, defined as < 4% of the level of the GPI (+) cell population, after gating for cells with at least 10% of the PE fluorescence of the GPI (+) population. For a negative control, we analyzed 2 non-malignant B-lymphoblastoid cell lines (BLCLs) from normal donors, and for a positive control, we analyzed the mantle cell lymphoma cell line HBL2A (in this case using CD45-PE to identify transmembrane proteins). The normal BLCLs demonstrated a frequency of PNH cells of 6.3 × 10−6 and 18.4 × 10−6, which is in the range that we have previously reported for BLCLs and granulocytes from normal individuals. These values are also similar to the frequency of spontaneously arising phenotypic variants in normal donors using other genes. In contrast, as we have previously reported, the mantle cell line demonstrated a markedly higher frequency of cells with the PNH phenotype– 1034 × 10−6. Of the involved marrow samples analyzed, there were at least 2 distinct groups. One group, representing 14 of the 20 samples (70%), demonstrated a mutant frequency that is comparable to non-malignant cell populations, with a median value of 9.5 × 10−6 (range 2.4 to 37 × 10−6). The remaining 6 samples (30%) demonstrated a markedly increased frequency of PNH cells, with a median value of 90 × 10−6 (range 73 to 11,763 × 10−6). Most of the samples analyzed were obtained from patients who had received prior therapy, but one of the samples demonstrating a very high frequency of PNH cells (1314 × 10−6) was derived from a patient who had not had prior therapy and was known to have had an abnormality of p53 based on FISH. This data demonstrates that an increase in inactivating mutations—as determined by this assay– is not essential for the development of myeloma, but it does seem to be a common feature of this condition. This flow-based assay could be applied at the time of diagnosis to facilitate investigations as to whether hypermutability correlates with outcome in patients with myeloma. Disclosures: No relevant conflicts of interest to declare.

Gain-of-Function KIT Mutations Sensitize the Mutant Isoform to the Type I Tyrosine Kinase Inhibitor Crenolanib: A Rationale for the Therapeutic Use in Systemic Mastocytosis (SM) and Core Binding Factor Leukemias (CBFL)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2230-2230 ◽  
Author(s):  
Marcus M Schittenhelm ◽  
Figen Akmut ◽  
Barbara Illing ◽  
Julia Frey ◽  
Katja Schuster ◽  
...  
Keyword(s):  
Mast Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Ex Vivo ◽  
Systemic Mastocytosis ◽  
Leukemia Cell ◽  
High Sensitivity ◽  
Hematologic Malignancies ◽  
Leukemia Cell Lines ◽  
Nanomolar Range

Abstract Activating mutations of the class III receptor tyrosine kinases FLT3 and KIT are associated with certain human neoplasms, including hematologic malignancies, i.e. the majority of patients with systemic mast cell disorders (KIT) and subsets of patients with acute myelogenous leukemia (FLT3 and KIT). Crenolanib is a potent selective FLT3 inhibitor with high efficacy against internal tandem dupliction mutations (ITD) – but also secondary kinase domain mutations conferring resistance towards other TKI. Interestingly, crenolanib does not target the wildtype KIT isoform, which is believed to reduce clinical side effects such as prolonged myelosuppression observed with other TKI. Clinical studies are currently enrolling. We now show that gain-of-function mutations of KIT, including codon D816 alterations as the most prevalent mutation in SM and CBFL, sensitize the mutant isoform to crenolanib. Several mast cell and leukemia cell lines harboring autoactivating KIT or FLT3 isoforms were treated with crenolanib in dose dilution series (MOLM14, MV4;11, HMC1.1/1.2, p815). To minimize cell-type specific off-target effects, an isogenic cell model was established. The murine pro B-cell line Ba/F3 was retrovirally transduced with either a FLT3 ITD or a KIT D816 isoform. Apoptosis induction was analyzed by annexin V-based assays. FLT3/KIT tyrosine phosphorylation was assessed by western immunoblots. As previously described, the FLT3 ITD positive cell line MOLM14 revealed high sensitivity towards crenolanib with IC50s in the lowest nanomolar range. We also confirmed high sensitivity towards crenolanib ex vivo in the low nanomolar range in a native sample of a heavily pretreated patient. This patient relapsed with FLT3 ITD positive leukemia harboring a secondary D835H mutation in a subclone. Interestingly, leukemia cells in the relapse situation were much more oncogene-addicted than cells at primary diagnosis, which is in line with previous findings by others. Due to the structural homology of FLT3 D835 and KIT D816 mutations, we extended our studies to mutant-KIT mastocytosis and leukemia cell models and confirm clinically relevant antiproliferative as well as proapoptotic sensitivities towards crenolanib: for HMC mastocytosis cells harboring a KIT V560G and/or a D816V mutation, potent induction of apoptosis was observed with IC50s of 100-250nM. The murine p815 mastocytosis cell line (harboring a D814Y mutation corresponding to D816Y in humans) demonstrated a proapoptotic effect of crenolanib with an IC50 of 60 nM. Treatment of corresponding KIT or FLT3 isoform-transduced Ba/F3 cells confirmed similar IC50s in the leukemia cell lines. Parental Ba/F3cells did not show any sensitivity towards crenolanib up to concentrations of 1000 nM. Additionally, potent dephosphorylation at 100 nM of KIT D816V in Ba/F3 and HMC cells after exposure to crenolanib confirmed mutant-KIT as a target of the drug. Evaluation of a broader range of native mast cell and leukemia patient samples as well as additional leukemia cell lines and isogenic Ba/F3 KIT or FLT3 transfectants is ongoing. First results demonstrate activity of crenolanib in native cells of a subset of patient samples with SM or CBFL treated ex vivo. Even more, combination of crenolanib with anthracyclines revealed additive to superadditive proapoptotic effects. Moreover, combination of crenolanib with cladribine, a hallmark agent in the treatment of systemic mastocytosis, resulted in potent induction of apoptosis already at doses that did not display any proapoptotic effects when administered as single agents, thereby providing a rationale for combinatorial therapeutic approaches. In summary, crenolanib is effective against the KIT D816V isoform associated with several hematologic malignancies. Notably, while not as effective towards mutant-KIT compared to the FLT3 ITD isoform, the observed estimated IC50 of crenolanib is well in the range of achievable plasma concentrations and in the range of the potent KIT inhibitor dasatinib, which is successfully under clinical investigation in CBFL. Our data provide a rationale to test crenolanib as a potent inhibitor of mutant-KIT isoforms in KIT-associated neoplasms. Disclosures Schuster: AROG Pharmaceuticals: Employment. Ramachandran:AROG: Employment.

scholarly journals Re-expression of ABP-120 rescues cytoskeletal, motility, and phagocytosis defects of ABP-120- Dictyostelium mutants.

1996 ◽  
Vol 7 (5) ◽  
pp. 803-823 ◽  
Author(s):  
D Cox ◽  
D Wessels ◽  
D R Soll ◽  
J Hartwig ◽  
J Condeelis
Keyword(s):  
Homologous Recombination ◽  
Cell Line ◽  
Cell Lines ◽  
Binding Proteins ◽  
Chemical Mutagenesis ◽  
Actin Binding ◽  
Direct Result ◽  
Cross Linking ◽  
Wild Type ◽  
Actin Binding Proteins

The actin binding protein ABP-120 has been proposed to cross-link actin filaments in nascent pseudopods, in a step required for normal pseudopod extension in motile Dictyostelium amoebae. To test this hypothesis, cell lines that lack ABP-120 were created independently either by chemical mutagenesis or homologous recombination. Different phenotypes were reported in these two studies. The chemical mutant shows only a subtle defect in actin cross-linking, while the homologous recombinant mutants show profound defects in actin cross-linking, cytoskeletal structure, pseudopod number and size, cell motility and chemotaxis and, as shown here, phagocytosis. To resolve the controversy as to what the ABP-120- phenotype is, ABP-120 was re-expressed in an ABP-120- cell line created by homologous recombination. Two independently "rescued" cell lines that express wild-type levels of ABP-120 were analyzed. In both rescued cell lines, actin incorporation into the cytoskeleton, pseudopod formation, cell morphology, instantaneous velocity, phagocytosis, and chemotaxis were restored to wild-type levels. There is no alteration in the expression levels of several related actin binding proteins in either the original ABP-120- cell line or in the rescued cell lines, leading to the conclusion that neither the aberrant phenotype observed in ABP-120- cells nor the normal phenotype reasserted in rescued cells can be attributed to alterations in the levels of other abundant and related actin binding proteins. Re-expression of ABP-120 in ABP-120- cells reestablishes normal structural and behavioral parameters, demonstrating that the severity and properties of the structural and behavioral defects of ABP-120- cell lines produced by homologous recombination are the direct result of the absence of ABP-120.

scholarly journals Combination Benefit of Capivasertib and Venetoclax in Preclinical Models of Diffuse Large B-Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1870-1870
Author(s):  
Brandon Willis ◽  
India Neveras ◽  
Hannah Dry ◽  
Wendan Xu ◽  
Yang Li ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Mouse Models ◽  
Cell Lines ◽  
Stock Options ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Tumor Growth Inhibition ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell malignancy among adults and despite approximately 65% of patients with DLBCL being cured with RCHOP therapy, nonresponsive and relapsed patients have inadequate treatment options, highlighting the importance for innovative treatment regimens. Blockade of B-cell receptor (BCR) downstream signaling components with various targeted agents is emerging as a clinically tractable treatment strategy across multiple B-cell malignancies. Protein Kinase B (AKT) signaling downstream of the BCR complex has been shown to be a central node in germinal center B-cell (GCB) DLBCL and the potent, selective inhibitor of AKT1, AKT2, AKT3, capivasertib, currently being evaluated in multiple clinical trials by targeting AKT-driven solid cancers, has been shown to induce apoptosis in a subset of GCB-DLBCL cell lines and cause tumor stasis in xenograft mouse models (Erdman et al., 2017). Since the monotherapy capivasertib responses in GCB DLBCL models are partial and lack durability, we hypothesized a combination approach could deliver even greater therapeutic benefit. To identify optimal partners, we conducted a capivasertib centric in vitro combination screen with specific with BH3 family members across a panel of 15 DLBCL cell lines, which revealed a synergistically active combination with the BCL2 inhibitor, venetoclax which is currently being evaluated in DLBCL. The activity was specifically enhanced in cell lines of the GCB subtype, with 4 PTEN del and 2 PTEN wt cell line models showing combination benefit. To determine the ability of this combination to drive stronger and durable responses, we assessed capivasertib and ventoclax activity in xenograft mouse models using two GCB-DLBCL cell line lines, SUDHL4 (PTEN wt) and WSU-DLCL2 (PTEN del). Oral administration of either monotherapy capivasertib (130 mg/kg BID, 4-day on/3-day off) or venetoclax (100 mg/kg QD) provided partial tumor growth inhibition (capivasertib TGI = 74% in SUDHL4 and 29% in WSU-DLCL2, and venetoclax TGI = 46% in SUDHL4 and 0% in WSU-DLCL2), whereas the combination of capivasertib and venetoclax both on a 4-day on/3-day off schedule produced complete tumor regression (100% regression) in both xenograft GCB cell line models during the dosing period. Notably, in both xenograft models all mice (5/5 per model) remained tumor free for at least 30 days following dosing cessation demonstrating high durability of response for the combination. Additionally, this combination is currently being evaluated in clinically relevant GCB and non-GCB PDX mouse models. Taken together, our results provide preclinical evidence for the rational combination of AKT and BCL-2 blockade with capivasertib and venetoclax respectively in patients with relapsed/refractory GCB-DLBCL. Disclosures Willis: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Neveras: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Dry: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Mongeon: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Rosen: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Mettetal: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Barry: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options.

scholarly journals Regulatory landscapes of specific miRNAs are conserved between cell lines and primary tumors

F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 633
Author(s):  
Hanwen Zhu ◽  
Boting Ning
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Mirna Expression ◽  
Regulatory Network ◽  
Human Cancer ◽  
Cancer Cell Line ◽  
Cancer Type ◽  
Cancer Tissues

Background: MicroRNAs are essential gene expression regulators and play important roles in various biological processes, such as cancer. They have shown great translational promise as either diagnostic biomarkers or therapeutic targets. While the similarities between transcriptomic profiles from The Cancer Genome Atlas and the Cancer Cell Line Encyclopedia have been thoroughly studied before, less is known on the microRNA side. This project aims to provide critical biological knowledge on the extent of consensus microRNA expression and regulation between cell line models and primary human tumors.  Method: First, we examined the similarity of miRNA expression profiles between CCLE cell lines and TCGA tumor samples for each cancer type. Next, we compared the expression of miRNAs associating the hallmarks of cancer pathways. Finally, we constructed miRNA-mRNA regulatory network for each cancer type and evaluated whether the regulatory role of each miRNA is conserved between cell lines and tumor samples.   Results: Our results indicate that, similar to gene expression, how well cancer cell line microRNA expression would capture the transcriptomic profile of human cancer tissues is greatly affected by the tumor type and purity. The cell-type composition for a cancer type also affects how accurately cancer cell lines could reflect the miRNA expression in tumor tissues. Furthermore, through network analysis, we show that certain microRNAs, not all, regulate the same set of target genes in both the cell line and human cancer tissues.  Conclusions: Through systematically comparing the miRNA expression profile and the regulatory network, our study highlights the biological differences between cell line and tumor samples and provides resources for future miRNA and cancer studies.

scholarly journals BET Proteins as Attractive Targets for Cancer Therapeutics

2021 ◽  
Vol 22 (20) ◽  
pp. 11102
Author(s):  
Joanna Sarnik ◽  
Tomasz Popławski ◽  
Paulina Tokarz
Keyword(s):  
Homologous Recombination ◽  
Transcriptional Control ◽  
Synthetic Lethality ◽  
Clinical Success ◽  
Predictive Biomarkers ◽  
Cancer Therapeutics ◽  
Parp Inhibitors ◽  
Novel Approach ◽  
Bet Inhibitors ◽  
Recombination Pathway

Transcriptional dysregulation is a hallmark of cancer and can be an essential driver of cancer initiation and progression. Loss of transcriptional control can cause cancer cells to become dependent on certain regulators of gene expression. Bromodomain and extraterminal domain (BET) proteins are epigenetic readers that regulate the expression of multiple genes involved in carcinogenesis. BET inhibitors (BETis) disrupt BET protein binding to acetylated lysine residues of chromatin and suppress the transcription of various genes, including oncogenic transcription factors. Phase I and II clinical trials demonstrated BETis’ potential as anticancer drugs against solid tumours and haematological malignancies; however, their clinical success was limited as monotherapies. Emerging treatment-associated toxicities, drug resistance and a lack of predictive biomarkers limited BETis’ clinical progress. The preclinical evaluation demonstrated that BETis synergised with different classes of compounds, including DNA repair inhibitors, thus supporting further clinical development of BETis. The combination of BET and PARP inhibitors triggered synthetic lethality in cells with proficient homologous recombination. Mechanistic studies revealed that BETis targeted multiple essential homologous recombination pathway proteins, including RAD51, BRCA1 and CtIP. The exact mechanism of BETis’ anticancer action remains poorly understood; nevertheless, these agents provide a novel approach to epigenome and transcriptome anticancer therapy.

scholarly journals CD38low Natural Killer Cells Transiently Expressing CD16F158V m-RNA Potentiates the Therapeutic Activity of Daratumumab Against Multiple Myeloma with Minimal Effector NK Cell Fratricide

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3199-3199 ◽  
Author(s):  
Subhashis Sarkar ◽  
Sachin Chauhan ◽  
Arwen Stikvoort ◽  
Alessandro Natoni ◽  
John Daly ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
Advisory Committees ◽  
Cd38 Expression ◽  
Rpmi 8226

Abstract Introduction: Multiple Myeloma (MM) is a clonal plasma cell malignancy typically associated with the high and uniform expression of CD38 transmembrane glycoprotein. Daratumumab is a humanized IgG1κ CD38 monoclonal antibody (moAb) which has demonstrated impressive single agent activity even in relapsed refractory MM patients as well as strong synergy with other anti-MM drugs. Natural Killer (NK) cells are cytotoxic immune effector cells mediating tumour immunosurveillance in vivo. NK cells also play an important role during moAb therapy by inducing antibody dependent cellular cytotoxicity (ADCC) via their Fcγ RIII (CD16) receptor. Furthermore, 15% of the population express a naturally occurring high affinity variant of CD16 harbouring a single point polymorphism (F158V), and this variant has been linked to improved ADCC. However, the contribution of NK cells to the efficacy of Daratumumab remains debatable as clinical data clearly indicate rapid depletion of CD38high peripheral blood NK cells in patients upon Daratumumab administration. Therefore, we hypothesize that transiently expressing the CD16F158V receptor using a "safe" mRNA electroporation-based approach, on CD38low NK cells could significantly enhance therapeutic efficacy of Daratumumab in MM patients. In the present study, we investigate the optimal NK cell platform for generating CD38low CD16F158V NK cells which can be administered as an "off-the-shelf"cell therapy product to target both CD38high and CD38low expressing MM patients in combination with Daratumumab. Methods: MM cell lines (n=5) (MM.1S, RPMI-8226, JJN3, H929, and U266) and NK cells (n=3) (primary expanded, NK-92, and KHYG1) were immunophenotyped for CD38 expression. CD16F158V coding m-RNA transcripts were synthesized using in-vitro transcription (IVT). CD16F158V expression was determined by flow cytometry over a period of 120 hours (n=5). 24-hours post electroporation, CD16F158V expressing KHYG1 cells were co-cultured with MM cell lines (n=4; RPMI-8226, JJN3, H929, and U266) either alone or in combination with Daratumumab in a 14-hour assay. Daratumumab induced NK cell fratricide and cytokine production (IFN-γ and TNF-α) were investigated at an E:T ratio of 1:1 in a 14-hour assay (n=3). CD38+CD138+ primary MM cells from newly diagnosed or relapsed-refractory MM patients were isolated by positive selection (n=5), and co-cultured with mock electroporated or CD16F158V m-RNA electroporated KHYG1 cells. CD16F158V KHYG1 were also co-cultured with primary MM cells from Daratumumab relapsed-refractory (RR) patients. Results: MM cell lines were classified as CD38hi (RPMI-8226, H929), and CD38lo (JJN3, U266) based on immunophenotyping (n=4). KHYG1 NK cell line had significantly lower CD38 expression as compared to primary expanded NK cells and NK-92 cell line (Figure 1a). KHYG1 electroporated with CD16F158V m-RNA expressed CD16 over a period of 120-hours post-transfection (n=5) (Figure 1b). CD16F158V KHYG1 in-combination with Daratumumab were significantly more cytotoxic towards both CD38hi and CD38lo MM cell lines as compared to CD16F158V KHYG1 alone at multiple E:T ratios (n=4) (Figure 1c, 1d). More importantly, Daratumumab had no significant effect on the viability of CD38low CD16F158V KHYG1. Moreover, CD16F158V KHYG1 in combination with Daratumumab produced significantly higher levels of IFN-γ (p=0.01) upon co-culture with CD38hi H929 cell line as compared to co-culture with mock KHYG1 and Daratumumab. The combination of CD16F158V KHYG1 with Daratumumab was also significantly more cytotoxic to primary MM cell ex vivo as compared to mock KHYG1 with Daratumumab at E:T ratio of 0.5:1 (p=0.01), 1:1 (p=0.005), 2.5:1 (p=0.003) and 5:1 (p=0.004) (Figure 1e). Preliminary data (n=2) also suggests that CD16F158V expressing KHYG1 can eliminate 15-17% of primary MM cells from Daratumumab RR patients ex vivo. Analysis of more Daratumumab RR samples are currently ongoing. Conclusions: Our study provides the proof-of-concept for combination therapy of Daratumumab with "off-the-shelf" CD38low NK cells transiently expressing CD16F158V for treatment of MM. Notably, this approach was effective against MM cell lines even with low CD38 expression (JJN3) and primary MM cells cultured ex vivo. Moreover, the enhanced cytokine production by CD16F158V KHYG1 cells has the potential to improve immunosurveillance and stimulate adaptive immune responses in vivo. Disclosures Sarkar: Onkimmune: Research Funding. Chauhan:Onkimmune: Research Funding. Stikvoort:Onkimmune: Research Funding. Mutis:Genmab: Research Funding; OnkImmune: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; Celgene: Research Funding; Novartis: Research Funding. O'Dwyer:Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; BMS: Research Funding; Glycomimetics: Research Funding; Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Matching cell lines with cancer type and subtype of origin via mutational, epigenomic and transcriptomic patterns

2019 ◽  
Author(s):  
Marina Salvadores ◽  
Francisco Fuster-Tormo ◽  
Fran Supek
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Experimental Work ◽  
Drug Sensitivity ◽  
Tissue Cell ◽  
Cancer Type ◽  
Tumor Type ◽  
Cell Type ◽  
Cancer Subtypes ◽  
The One

AbstractCell lines are commonly used as cancer models. Because the tissue and/or cell type of origin provide important context for understanding mechanisms of cancer, we systematically examined whether cell lines exhibit features matching the cancer type that supposedly originated them. To this end, we aligned the mRNA expression and DNA methylation data between ∼9,000 solid tumors and ∼600 cell lines to remove the global differences stemming from growth in cell culture. Next, we created classification models for cancer type and subtype using tumor data, and applied them to cell line data. Overall, the transcriptomic and epigenomic classifiers consistently identified 35 cell lines which better matched a different tissue or cell type than the one the cell line was originally annotated with; we recommend caution in using these cell lines in experimental work. Six cell lines were identified as originating from the skin, of which five were further corroborated by the presence of a UV-like mutational signature in their genome, strongly suggesting mislabelling. Overall, genomic evidence additionally supports that 22 (3.6% of all considered) cell lines may be mislabelled because we predict they originate from a different tissue/cell type. Finally, we cataloged 366 cell lines in which both transcriptomic and epigenomic profiles strongly resemble the tumor type of origin, designating them as ‘golden set’ cell lines. We suggest these cell lines are better suited for experimental work that depends on tissue identity and propose tentative assignments to cancer subtypes. Finally, we show that accounting for the uncertain tissue-of-origin labels can change the interpretation of drug sensitivity and CRISPR genetic screening data. In particular, in brain, lung and pancreatic cancer cell lines, many novel determinants of drug sensitivity or resistance emerged by focussing on the cell lines that are best matched to the cancer type of interest.

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