The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor–armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms. Significance CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.

We demonstrate how mutant and wild-type DNA sequences result in different pyrograms. Using pyrograms of established mutations in tumors, we explain how to analyze the pyrogram peaks generated by different dispensation sequences. Further, we demonstrate some limitations of pyrosequencing, including how some complex mutations can be indistinguishable from single base mutations. Pyrosequencing is the basis of the Roche 454 next-generation sequencer and many of the same principles also apply to the Ion Torrent hydrogen ion-based next-generation sequencers.

BACKGROUND & AIMS Pancreatic imaging can identify neoplastic cysts but not microscopic neoplasms. Mutation analysis of pancreatic fluid following secretin stimulation might identify microscopic neoplasias in the pancreatic duct system. We determined the prevalence of mutations in KRAS and GNAS genes in pancreatic juice from subjects undergoing endoscopic ultrasound for suspected pancreatic intraepithelial neoplasia (PanIN), intraductal papillary mucinous neoplasms, or pancreatic adenocarcinoma. METHODS Secretin-stimulated juice samples were collected from the duodenum of 272 subjects enrolled in Cancer of the Pancreas Screening studies; 194 subjects were screened because of a family history of, or genetic predisposition to, pancreatic cancer and 78 were evaluated for pancreatic cancer (n=30) or other disorders (controls: pancreatic cysts, pancreatitis, or normal pancreata, n=48). Mutations were detected by digital high-resolution melt-curve analysis and pyrosequencing. The number of replicates containing a mutation determined the mutation score. RESULTS KRAS mutations were detected in pancreatic juice from larger percentages of subjects with pancreatic cancer (73%) or undergoing cancer screening (50%) than controls (19%) (P=.0005). A greater proportion of patients with pancreatic cancer had at least 1 KRAS mutation detected 3 or more times (47%) than screened subjects (21%) or controls (6%, P=.002). Among screened subjects, mutations in KRAS (but not GNAS) were found in similar percentages of patients with or without pancreatic cysts. However, a greater proportion of patients over 50 ys old had KRAS mutations (54.6%) than younger patients (36.3%) (P=.032); the older subjects also more mutations in KRAS (P=.02). CONCLUSIONS Mutations in KRAS are detected in pancreatic juice from the duodenum of 73% of patients with pancreatic cancer, and 50% of asymptomatic individuals with a high risk for pancreatic cancer. However, KRAS mutations are detected in pancreatic juice from 19% of controls. Mutations detected in individuals without pancreatic abnormalities, based on imaging analyses, likely arise from small PanIN lesions. ClinicalTrials.gov no: NCT00438906 and NCT00714701

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Therapeutic targeting of initiating oncogenes is the mainstay of precision medicine. Considerable efforts have been expended toward silencing MYC, which drives many human cancers including Burkitt lymphomas (BL). Yet, the effects of MYC silencing on standard-of-care therapies are poorly understood. Here we found that inhibition of MYC transcription renders B-lymphoblastoid cells refractory to chemotherapeutic agents. This suggested that in the context of chemotherapy, stabilization of Myc protein could be more beneficial than its inactivation. We tested this hypothesis by pharmacologically inhibiting glycogen synthase kinase 3β (GSK-3β), which normally targets Myc for proteasomal degradation. We discovered that chemorefractory BL cell lines responded better to doxorubicin and other anti-cancer drugs when Myc was transiently stabilized. In vivo, GSK3 inhibitors (GSK3i) enhanced doxorubicin-induced apoptosis in BL patient-derived xenografts (BL-PDX), as well as in murine MYC-driven lymphoma allografts. This enhancement was accompanied by and required deregulation of several key genes acting in the extrinsic, death-receptor-mediated apoptotic pathway. Consistent with this mechanism of action, GSK3i also facilitated lymphoma cell killing by a death ligand TRAIL and by a death receptor agonist mapatumumab. Thus, GSK3i synergizes with both standard chemotherapeutics and direct engagers of death receptors and could improve outcomes in patients with refractory lymphomas.Transient stabilization, rather than inhibition, of MYC amplifies extrinsic apoptosis and therapeutic. . .

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Objectives BRAF mutations have substantial therapeutic, diagnostic, and prognostic significance, so detecting and specifying them is an important part of the workload of molecular pathology laboratories. Pyrosequencing assays are well suited for this analysis but can produce complex results. Therefore, we introduce a pyrosequencing lookup table based on Pyromaker that assists the user in generating hypotheses for solving complex pyrosequencing results. Methods The lookup table contains all known mutations in the sequenced region and the positions in the dispensation sequence at which changes would occur with those mutations. We demonstrate the lookup table using a homebrew dispensation sequence for BRAF codons 596 to 605 as well as a commercially available kit-based dispensation sequence for codons 599 to 600. Results These results demonstrate that the homebrew dispensation sequence unambiguously identifies all known BRAF mutations in this region, whereas the kit-based dispensation sequence has one unresolvable degeneracy that could be solved with the addition of two injections. Conclusions Using the lookup table and confirmatory virtual pyrogram, we unambiguously solved clinical pyrograms of the complex mutations V600K (c.1798_1799delGTinsAA), V600R (c.1798_1799delGTinsAG), V600D (c.1799_1800delTGinsAT), V600E (c.1799_1800delTGinsAA), and V600_K601delinsE (c.1799_1801delTGA). In addition, we used the approach to hypothesize and confirm a new mutation in human melanoma, V600_K601delinsEI (c.1799_1802delTGAAinsAAAT).

The Myc proto-oncogene is known to regulate p53-dependent apoptosis through the Myc -> ARF -| MDM2 -| p53 pathway. We had previously shown that this pathway could be exploited to increase sensitivity to anti-cancer drugs. Specifically, small increases in Myc levels obtained by inhibiting the Myc-targeting microRNA-34a resulted in enhanced apoptotic response of B-cells to bortezomib (Sotillo et al, Oncogene 2010). However, many Myc-driven tumors are deficient in p53 activity. Thus, we sought to determine whether transient up-regulation of Myc also increases cell death in response to chemotherapeutic drugs in p53-deficient backgrounds. We chose to study this in human Burkitt's lymphoma cell lines and murine B-lymphoid cells isolated from bone marrows of p53ER™ knock-in mice and subsequently transduced with a Myc-expressing retrovirus (Amaravadi et al, J Clin Inv 2007, Yu et al, Blood 2007). This latter system allowed us to distinguish between p53-dependent (cells treated with tamoxifen) and independent (no tamoxifen added) cell death. We also considered that Myc expression is regulated not only transcriptionally, most notably by the bromodomain containing protein 4 (BRD4), but also post-transcriptionally by the glycogen synthase kinase 3β (GSK3-β). GSK3-β phosphorylates Myc on its Thr58 residue, targeting it for proteasomal degradation, and inhibition of GSK3-β activity results in stabilization of Myc protein (Chung et al, J Clin Inv 2012). Using the GSK3-β inhibitor CHIR 99021, we transiently increased Myc protein levels prior to treating B-lymphoma cells with chemotherapeutic drugs. Remarkably, pharmacological stabilization of Myc strongly enhanced p53-independent doxorubicin-induced apoptosis in the p53ER™/Myc system as well as in Burkitt's lymphoma cell lines with inactive p53. This enhancement was accompanied by cleavage of caspase 8 with concomitant de-regulation of the death receptor pathway genes, indicating engagement of the extrinsic apoptotic pathway. In contrast, we did not observe any significant differences in the intrinsic pro-apoptotic genes (BAX, BAK, BIM). To determine whether chemosensitization was mediated by Myc or other GSK3-β targets, we used a BRD4 inhibitor (iBET-151) at concentrations that were sufficient to prevent the CHIR 99021-mediated increase in Myc levels but didn't grossly affect cell viability. Inhibition of Myc with iBET-151 abrogated the increase in apoptosis brought about by CHIR 99021 and doxorubicin, suggesting that Myc is key to chemosensitization by CHIR 99021. Our results suggest that Myc stabilization could be a viable adjuvant therapy, even for tumors with p53 loss or MDM2 amplification. Citation Format: Elena Sotillo-Piñeiro, Colleen T. Harrington, Daniel Soto De Jesus, Andrei Thomas-Tikhonenko. Transient upregulation of Myc with GSK3-β inhibitors in B-cell lymphomas enhances p53-independent apoptotic responses to chemotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B33.