Growth Advantage In Culture Research Articles

Abstract PO-32: NFKBIZ 3′ UTR mutations confer selective growth advantage and affect drug response in diffuse large B-cell lymphoma

Abstract Introduction: The activated B cell-like (ABC) molecular subgroup of diffuse large B-cell lymphoma (DLBCL) is characterized by activation of NF-κB signaling. Recurrent mutations affecting genes such as MYD88, CD79A/B, and TNFAIP3 contribute to this in some cases, but there remain tumors with no known genetic basis for this pathway activation. This suggests that our understanding of ABC DLBCL drivers remains incomplete. Previously, NFKBIZ was shown to be amplified in 10% of ABC DLBCLs and to contribute to activation of NF-κB signaling. We recently described a novel pattern of mutations affecting the 3′ UTR of NFKBIZ resulting in an overall mutation rate of 30% (UTR or AMP) in ABC DLBCL. These NFKBIZ UTR mutations are mutually exclusive with MYD88 mutations, thus suggesting they may also lead to activation of NF-κB signaling. The NFKBIZ protein interacts with NF-κB transcription factors and is thought to regulate canonical NF-κB signaling. We hypothesized that NFKBIZ UTR mutations affect the normally rapid degradation of this mRNA by disrupting secondary structures recognized by RNA-binding proteins such as ribonucleases. The resulting elevated NFKBIZ mRNA levels would lead to accumulation of protein and may be a novel mechanism to promote cell growth and survival in ABC DLBCL. Methods: NFKBIZ 3′ UTR mutations were introduced into a DLBCL cell line using the CRISPR-Cas9 system. A competitive growth assay with wild-type (WT) and CRISPR-mutant lines was performed to assess whether UTR mutations provide a growth advantage in culture (in vitro) and in mouse xenografts (in vivo). RNA-sequencing was then performed on WT and a subset of CRISPR-mutant lines and analyses were performed to identify genes upregulated by IκB-ζ in mutant lines. The IC50 of relevant drugs was determined by WST-1 assays after drug treatment on WT and mutant lines. Results: Introduction of NFKBIZ mutations into a DLBCL cell line confirmed that UTR deletions lead to increased mRNA and protein levels. NFKBIZ UTR deletions give DLBCL cells a selective growth advantage over WT both in vitro and in vivo. RNA-sequencing of mutant and WT lines revealed possible transcriptional targets of NFKBIZ, including NF-κB targets and genes commonly overexpressed in ABC DLBCL. Novel candidate NFKBIZ targets were also discovered through this analysis, including CD274, the gene encoding PD-L1. Mutant cell lines had significantly higher IC50 compared to WT for the drugs ibrutinib, idelalisib, and masitinib, but not bortezomib, suggesting that NKFBIZ UTR mutations make cell lines more resistant to specific NF-κB pathway-targeted drugs. Conclusions: This work directly establishes a role for NFKBIZ amplifications and 3′ UTR mutations in driving ABC DLBCL through NF-κB signaling. We demonstrate that these mutations can lead to overexpression of NFKBIZ and provide a selective growth advantage to cells both in vitro and in vivo. In addition, we found that these mutant lines were more resistant to some targeted lymphoma drugs but not others. Citation Format: Sarah E. Arthur, Nicole Thomas, Jeffrey Tang, Christopher K. Rushton, Miguel Alcaide, Adèle Telenius, Shannon Healy, Anja Mottok, David W. Scott, Christian Steidl, Ryan D. Morin. NFKBIZ 3′ UTR mutations confer selective growth advantage and affect drug response in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-32.

NFKBIZ3′ UTR Mutations Confer Selective Growth Advantage and Activate Genes with Therapeutic Implications in Diffuse Large B-Cell Lymphoma

Introduction: The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) is characterized by activation of NF-κB signaling and increased mortality. Recurrent mutations affecting genes such as MYD88, CD79A/B and TNFAIP3 have been shown to be involved in activation of the NF-κB pathway in ABC DLBCL; however there still remain cases with no known genetic basis for this pathway activation suggesting that our understanding of the drivers of ABC DLBCL remains incomplete. Previously, NFKBIZ was shown to be amplified in ~10% of ABC DLBCLs and to contribute to activation of NF-κB signaling. We have recently described a novel pattern of mutations affecting the 3′ UTR of NFKBIZ resulting in an overall mutation rate of 34% for this gene (UTR mutations or amplifications) in ABC DLBCL. These NFKBIZ UTR mutations are mutually exclusive with MYD88 mutations, thus suggesting they may also lead to activation of NF-κB signaling. NFKBIZ encodes the IκB-ζ protein, which interacts with NF-κB transcription factors and is thought to regulate canonical NF-κB signaling. We hypothesized that NFKBIZ UTR mutations affect the normally rapid degradation of mRNA by disrupting secondary structures recognized by RNA-binding proteins such as ribonucleases. The resulting elevated mRNA levels would in turn lead to accumulation of IκB-ζ protein as a novel mechanism to promote cell growth and survival in ABC DLBCL. Methods: NFKBIZ 3′ UTR mutations were introduced into a DLBCL cell line (WSU-DLCL2) using the CRISPR-Cas9 system. NFKBIZ mRNA and protein levels were evaluated using custom designed droplet digital PCR assays and western blot analyses. Cells were stimulated with LPS to induce NFKBIZ expression and mRNA and protein levels were measured in wild-type (WT) and CRISPR-mutant lines to compare rates of mRNA decay and protein expression. A competitive growth assay with WT and CRISPR-mutant lines was performed to assess whether UTR mutations provide a growth advantage in culture (in vitro) and in mouse xenografts (in vivo). The composition of the pool of WT and mutant lines was determined by comparing WT and mutant DNA sequence proportions. RNA-sequencing was then performed on WT and a subset of CRISPR-mutant cell lines to identify genes up-regulated by IκB-ζ in mutant lines. Findings from these models were compared to the effects of NFKBIZ over-expression in patient tissues. Results: Introduction of NFKBIZ mutations into a DLBCL cell line confirmed that UTR deletions lead to increased mRNA and protein levels. LPS stimulation showed prolonged mRNA elevation in mutant lines, consistent with our model wherein these mutations disrupt post-transcriptional regulatory mechanisms. NFKBIZ UTR deletions gave DLBCL cells a selective growth advantage over WT both in vitro (cell culture) and in vivo (xenograft mouse model). RNA-sequencing of mutant and WT lines revealed possible transcriptional targets of IκB-ζ including some NF-κB targets and genes commonly over-expressed in ABC DLBCL (BATF, MAML2, and TNFRSF13B). HCK, a gene known to be activated by MYD88 was also upregulated in NFKBIZ mutant lines. This is consistent with our hypothesis that mutations in MYD88 and the NFKBIZ UTR are mutually exclusive because they activate similar pathways. HCK is also a target of ibrutinib, suggesting the potential utility of ibrutinib in these patients. Novel targets of IκB-ζ were also discovered through this analysis including CD274, the gene encoding PD-L1. This could be a novel mechanism for DLBCL tumours to express PD-L1 and therefore suggest that these tumours may be susceptible to anti-PD1/PD-L1 immunotherapies. Conclusions: This work highlights the role of NFKBIZ and 3′ UTR mutations in driving ABC DLBCL. We demonstrate that these mutations can lead to over-expression of NFKBIZ and provide a selective growth advantage to cells both in vitro and in vivo. In addition, we described multiple targets of IκB-ζ that may have implications in treatment susceptibility and/or resistance in ABC DLBCL. These findings contribute to a better understanding of the genetic basis of DLBCL, which is necessary to guide personalized therapeutic strategies. Disclosures Scott: Roche/Genentech: Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding; Janssen: Consultancy, Research Funding; Celgene: Consultancy. Steidl:Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Juno Therapeutics: Consultancy; Tioma: Research Funding; Bayer: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy.

Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations.

Human pluripotent stem cells (hPSCs) can self-renew indefinitely, making them an attractive source for regenerative therapies. This expansion potential has been linked with acquisition of large copy number variants (CNVs) that provide mutant cells with a growth advantage in culture1–3. However, the nature, extent, and functional impact of other acquired genome sequence mutations in cultured hPSCs is not known. Here, we sequenced the protein-coding genes (exomes) of 140 independent human embryonic stem cell (hESC) lines, including 26 lines prepared for potential clinical use4. We then applied computational strategies for identifying mutations present in a subset of cells5. Though such mosaic mutations were generally rare, we identified five unrelated hESC lines that carried six mutations in the TP53 gene that encodes the tumor suppressor P53. Notably, the TP53 mutations we observed are dominant negative and are the mutations most commonly seen in human cancers. We used droplet digital PCR to demonstrate that the TP53 mutant allelic fraction increased with passage number under standard culture conditions, suggesting that P53 mutation confers selective advantage. When we then mined published RNA sequencing data from 117 hPSC lines, we observed another nine TP53 mutations, all resulting in coding changes in the DNA binding domain of P53. Strikingly, in three lines, the allelic fraction exceeded 50%, suggesting additional selective advantage resulting from loss of heterozygosity at the TP53 locus. As the acquisition and favored expansion of cancer-associated mutations in hPSCs may go unnoticed during most applications, we suggest that careful genetic characterization of hPSCs and their differentiated derivatives should be carried out prior to clinical use.

Acquired Genetic and Epigenetic Variation in Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) can acquire non-random genomic variation during culture. Some of these changes are common in tumours and confer a selective growth advantage in culture. Additionally, there is evidence that reprogramming of human induced pluripotent stem cells (hiPSCs) introduces mutations. This poses a challenge to both the safety of clinical applications and the reliability of basic research using hPSCs carrying genomic variation. A number of methods are available for monitoring the genomic integrity of hPSCs, and a balance between practicality and sensitivity must be considered in choosing the appropriate methods for each use of hPSCs. Adjusting protocols by which hPSCs are derived and cultured is an evolving process that is important in minimising acquired genomic variation. Assessing genetic variation for its potential impact is becoming increasingly important as techniques to detect genome-wide variation improve.

GRK2 negatively regulates IGF‐1R signaling pathway and cyclins' expression in HepG2 cells

G protein coupled receptor kinase 2 (GRK2) plays a central role in the regulation of a variety of important signaling pathways. Alternation of GRK2 protein level and activity casts profound effects on cell physiological functions and causes diseases such as heart failure, rheumatoid arthritis, and obesity. We have previously reported that overexpression of GRK2 has an inhibitory role in cancer cell growth. To further examine the role of GRK2 in cancer, in this study, we investigated the effects of reduced protein level of GRK2 on insulin-like growth factor 1 receptor (IGF-1R) signaling pathway in human hepatocellular carcinoma (HCC) HepG2 cells. We created a GRK2 knockdown cell line using a lentiviral vector mediated expression of GRK2 specific short hairpin RNA (shRNA). Under IGF-1 stimulation, HepG2 cells with reduced level of GRK2 showed elevated total IGF-1R protein expression as well as tyrosine phosphorylation of receptor. In addition, HepG2 cells with reduced level of GRK2 also demonstrated increased tyrosine phosphorylation of IRS1 at the residue 612 and increased phosphorylation of Akt, indicating a stronger activation of IGF-1R signaling pathway. However, HepG2 cells with reduced level of GRK2 did not display any growth advantage in culture as compared with the scramble control cells. We further detected that reduced level of GRK2 induced a small cell cycle arrest at G2/M phase by enhancing the expression of cyclin A, B1, and E. Our results indicate that GRK2 has contrasting roles on HepG2 cell growth by negatively regulating the IGF-1R signaling pathway and cyclins' expression.

NPM-ALK Mutants in Kinase Domain Exhibit Altered Kinase Activity and Various Sensitivity to ALK Inhibitors.

Abnormal expression of constitutively active anaplastic lymphoma kinase chimeric proteins (NPM-ALK) in the pathogenesis of anaplastic large-cell lymphoma (ALCL) is well defined. Recently, small molecule inhibitors have been reported that provide solid proof-of-concept validation that inhibition of ALK is sufficient to attenuate the growth and proliferation of ALK+ALCL cells. Although robust clinical response of ALCL patients to the ALK inhibitors is expected, some of those patients are also anticipated to develop resistance to an ALK inhibitor, most likely associated with single point mutations in the kinase domain of ALK. In this study, the nonsense mutants of NPM-ALK on the phosphate anchor region and the gatekeeper region were generated by site-directed mutagenesis and their kinase activity was measured in cells. NPM-ALK/L182M, L182V and L256M mutants displayed comparable or higher kinase activity in cells relative to NPM-ALK wt, and were able to render BaF3 cells into growth factor-independent growth, while NPM-ALK/L182R, L256R, L256V, L256P and L256Q displayed much weaker or little kinase activity in cells and failed to transform BaF3 cells. While NPM-ALK/L182M and L182V displayed comparable sensitivity to a fused pyrrolocarbazole (FP)-derived ALK inhibitor as NPM-ALK wt, they were >30-fold less sensitive to a diaminopyrimidine (DAP)-derived ALK inhibitor. On the other hand, NPM-ALK/L256M displayed >30-fold less sensitivity to both the FP-derived and the DAP-derived ALK inhibitors. Consistent with NPM-ALK autophosphorylation inhibition, the FP-ALK inhibitor induced growth inhibition and cytotoxicity of BaF3/NPM-ALK/L182M and L182V cells but not L256M cells, and the DAP ALK inhibitor failed to induce growth inhibition and cytotoxicity of all three BaF3/NPM-ALK mutant cell lines in culture. In the absence of an ALK inhibitor, BaF3 cells harboring NPM-ALK mutants did not display growth advantage in culture with 10% serum and in mice over the NPM-ALK wt cells. However, the BaF3/NPM-ALK mutants displayed significant growth advantage in culture with low serum (2%) over the BaF3/NPM-ALKwt cells, suggesting the BAF3/NPM-ALK mutant cells may proliferate better in the stressed conditions. Analyses of binding of ALK inhibitors to ALK wt and mutants in ALK homology models are in progress and the results will be discussed.

Ectopic Activity of Fibroblast Growth Factor Receptor 1 in Hepatocytes Accelerates Hepatocarcinogenesis by Driving Proliferation and Vascular Endothelial Growth Factor–Induced Angiogenesis

Fibroblast growth factor (FGF) signaling mediates cell-to-cell communication in development and organ homeostasis in adults. Of the four FGF receptor (FGFR) tyrosine kinases, only FGFR4 is expressed in mature hepatocytes. Although FGFR1 is expressed by hepatic cell progenitors and adult nonparenchymal cells, ectopic expression is commonly observed in hepatoma cells. Here, we determined whether ectopic FGFR1 is a cause or consequence of hepatocellular carcinoma by targeting a constitutively active human FGFR1 to mouse hepatocytes. Livers of transgenic mice exhibited accelerated regeneration after partial hepatectomy but no signs of neoplastic or preneoplastic abnormalities for up to 18 months. However, in diethylnitrosamine-treated mice, the chronic FGFR1 activity promoted an incidence of 44% adenomas at 4 months and 38% hepatocellular carcinoma at 8 months. No adenoma or hepatocellular carcinoma was observed in diethylnitrosamine-treated wild-type (WT) livers at 4 or 8 months, respectively. At 10 and 12 months, tumor-bearing livers in transgenic mice were twice the size of those in WT animals. Isolated hepatoma cells from the transgenic tumors exhibited a growth advantage in culture. Advanced hepatocellular carcinoma in the transgenic livers exhibited a reduced rate of necrosis. This was accompanied by a mean microvessel density of 2.7 times that of WT tumors and a markedly higher level of vascular endothelial growth factor. In cooperation with an initiator, the persistent activity of ectopic FGFR1 in hepatocytes is a strong promoter of hepatocellular carcinoma by driving cell proliferation at early stages and promoting neoangiogenesis at late stages of progression.

Proteomic Analysis Reveals Hyperactivation of the Mammalian Target of Rapamycin Pathway in Neurofibromatosis 1–Associated Human and Mouse Brain Tumors

Individuals with the tumor predisposition syndrome, neurofibromatosis 1 (NF1), are prone to development of nervous system tumors, including neurofibromas and pilocytic astrocytomas. Based on the ability of the NF1 gene product (neurofibromin) to function as a GTPase activating protein for RAS, initial biologically based therapies for NF1-associated tumors focused on the use of RAS inhibitors, but with limited clinical success. In an effort to identify additional targets for therapeutic drug design in NF1, we used an unbiased proteomic approach to uncover unanticipated intracellular signaling pathways dysregulated in Nf1-deficient astrocytes. We found that the expression of proteins involved in promoting ribosome biogenesis was increased in the absence of neurofibromin. In addition, Nf1-deficient astrocytes exhibit high levels of mammalian target of rapamycin (mTOR) pathway activation, which was inhibited by blocking K-RAS or phosphatidylinositol 3-kinase activation. This mTOR pathway hyperactivation was reflected by high levels of ribosomal S6 activation in both Nf1 mutant mouse optic nerve gliomas and in human NF1-associated pilocytic astrocytoma tumors. Moreover, inhibition of mTOR signaling in Nf1-/- astrocytes abrogated their growth advantage in culture, restoring normal proliferative rates. These results suggest that mTOR pathway inhibition may represent a logical and tractable biologically based therapy for brain tumors in NF1.

Aberrations involving 13q12∼q14 are frequent secondary events in childhood acute lymphoblastic leukemia

Chromosome 13 aberrations, particularly in the region 13q12∼q14, are common events in hematological neoplasms that have a clinical significance in many cases. However, although chromosome 13 aberrations are a nonrandom event in childhood acute lymphoblastic leukemia (ALL), their biological and clinical associations are limited. We have studied a consecutive series of 277 cases of childhood ALL, including 33 initially at relapse, by conventional cytogenetic analysis. In 20 cases, a chromosome 13 aberration that involved the region 13q12∼q14 was detected at some point during the disease. An aberration was identified in 15 of 244 (6.1%) presentation cases and 7 of 54 (13%) relapsed cases, of which in 11 cases it was shown that the abnormality arose as a secondary karyotypic event. To further characterize the cases, fluorescence in situ hybridization (FISH) using the commercially available probes LSI 13 (RB1) and D13S25 (both 13q14) was undertaken. These analyses provided additional evidence for the secondary nature of many events and suggested that 13q14 deletions may confer a growth advantage in culture because the percentage of cells containing 13q14 deletions detected by FISH was often lower than that detected by G-banding. Since 13q12∼q14 aberrations in childhood ALL frequently occur as secondary events, these results imply that the abnormality has implications for disease progression.

γc Gene Transfer Into SCID X1 Patients’ B-Cell Lines Restores Normal High-Affinity Interleukin-2 Receptor Expression and Function

SCID X1 is characterized by faulty T-cell and natural killer cell differentiation caused by mutation of the gamma-c chain gene encoding a number of multiple cytokine receptors (interleukin-2 [IL-2], IL-4, IL-7, IL-9, and IL-15 receptors). To assess the feasibility of inducing long-term expression and function of the gamma-c chain, Epstein-Barr virus (EBV)-transformed B-cell lines from two patients with SCID X1 were transduced with a Moloney-derived retroviral vector containing the gamma-c chain cDNA. The viral LTR was used as the promoter. Immediately after two cycles of coculture with the psi-crip clone producing the MFG(B2)-gamma-c cDNA vector, gamma-c expression, assessed by detection of the mRNA and membrane protein expression, was found in 15% to 20% of cells. The degree of membrane expression was similar to that in control EBV-B cells. Expression increased steadily over 6 months, becoming detectable in 100% of cells, and remained stable thereafter for a total of 9 months, reflecting positive selection of transduced cells. A study of provirus integration sites showed multiple integration. The expressed gamma-c was functional, because it restored high-affinity IL-2 receptor binding, IL-2 endocytosis, and IL-2-triggered phosphorylation of JAK-3 tyrosine kinase. Similar results were obtained with the two B-cell lines. These results show that efficient gamma-c gene transfer into B-cells lacking functional gamma-c is feasible and results in strong and stable expression of a functional gamma-c chain, apparently conferring a selective growth advantage in culture. Further in vitro studies of gamma-c gene transfer into gamma-c- hematopoietic progenitors are being conducted to assess the feasibility of correcting lymphocyte differentiation defects.

HTLV-I infection of T and B cells of a patient with adult T-cell leukemia-lymphoma (ATLL) and transmission of HTLV-I from B cells to normal T cells

We analysed the DNA of different tissues of a patient (HS) with adult T-cell leukemia/lymphoma virus (HTLV-I). We detected viral sequences in fresh specimens from spleen, thymus, liver, skin and peripheral blood neoplastic lymphocytes. The pattern of HTLV-I intergration is identical in the leukemic cells and in all other tissues analysed, but the signal intensity is strongest in the leukemic cells, indicating the source of HTLV-I proviral sequences was the leukemic T-cells which had infiltrated these tissues. In fact, the cultured skin fibroblasts of the patient did not contain HTLV-I sequence. However, cultured lymphocytes of this patient was consistently an immortalized B-cell line containing HTLV-I sequences in a manner indicative of a polyclonal infection. This cell line was also infected with the Epstein-Barr virus (EBV). In order to determine whether HTLV-I alone was sufficient for B-cell immortalization, we obtained single cell clones by limiting dilution. The DNA of all the cell clones that we analysed contained both the HTLV-I and EBV genomes, suggesting that immortalization of the B-cell was more likely due to the EBV rather than HTLV-I. Infectious HTLV-I viruses produced by the B-cell line still had the propensity to infect and transform T-lymphocytes in normal human umbilical cord blood. Unlike the parental B cells, the transformed T lymphocytes were clonally selected. Our results indicate that although the predominant infected cell population of the patient was his leukemic T lymphocytes, some of his EBV-positive B-lymphocytes were also polyclonally infected. The latter had a growth advantage in culture over the T lymphocytes but the virus produced by these immortalized B cells has not been adapted and has maintained its tropism for T cells.