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Background and Aims: Isocitrate dehydrogenase 1 ( IDH1 )-mutant cholangiocarcinoma (CCA) is a highly lethal subtype of hepatobiliary cancer that is often resistant to immune checkpoint inhibitor therapies. We evaluated the effects of IDH1 mutations in CCA cells on the tumor immune microenvironment and identified opportunities for therapeutic intervention., Approach and Results: Analysis of 2606 human CCA tumors using deconvolution of RNA-sequencing data identified decreased CD8+ T cell and increased M2-like tumor-associated macrophage (TAM) infiltration in IDH1 -mutant compared to IDH1 wild-type tumors. To model the tumor immune microenvironment of IDH1 -mutant CCA in vivo, we generated an isogenic cell line panel of mouse SB1 CCA cells containing a heterozygous IDH1 R132C (SB1 mIDH1 ) or control (SB1 WT ) mutation using CRISPR-mediated homology-directed repair. SB1 mIDH1 cells recapitulated features of human IDH1 -mutant CCA including D-2-hydroxyglutarate production and increased M2-like TAM infiltration. SB1 mIDH1 cells and tumors produced increased levels of CCL2, a chemokine involved in the recruitment and polarization of M2-like TAMs, compared to wild-type controls. In vivo neutralization of CCL2 led to decreased M2-like TAM infiltration, reduced tumor size, and improved overall survival in mice harboring SB1 mIDH1 tumors., Conclusions: IDH1- mutant CCA is characterized by an increased abundance of M2-like TAMs. Targeting CCL2 remodels the tumor immune microenvironment and improves outcomes in preclinical models of IDH1 -mutant CCA, highlighting the role of myeloid-targeted immunotherapies in the treatment of this cancer., (Copyright © 2024 American Association for the Study of Liver Diseases.)

Abstract: Among the most common genetic alterations in myelodysplastic syndromes (MDS) are mutations in the spliceosome gene SF3B1. Such mutations induce specific RNA missplicing events, directly promote ring sideroblast (RS) formation, and generally associate with a more favorable prognosis. However, not all SF3B1 mutations are the same, and little is known about how distinct hotspots influence disease. Here, we report that the E592K variant of SF3B1 associates with high-risk disease features in MDS, including a lack of RS, increased myeloblasts, a distinct comutation pattern, and a lack of favorable survival seen with other SF3B1 mutations. Moreover, compared with other hot spot SF3B1 mutations, E592K induces a unique RNA missplicing pattern, retains an interaction with the splicing factor SUGP1, and preserves normal RNA splicing of the sideroblastic anemia genes TMEM14C and ABCB7. These data have implications for our understanding of the functional diversity of spliceosome mutations, as well as the pathobiology, classification, prognosis, and management of SF3B1-mutant MDS., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Identification of genomic signatures with consistent clinicopathological features in myelodysplastic/myeloproliferative neoplasm (MDS/MPN) is critical for improved diagnosis, elucidation of biology, inclusion in clinical trials, and development of therapies. We describe clinical and pathological features with co-existence of mutations in ASXL1 (missense or nonsense), SRSF2, and SKI homologous region of SETBP1, in 18 patients. Median age was 68 years with a male predominance (83%). Leukocytosis and neutrophilia were common at presentation. Marrow features included hypercellularity, granulocytic hyperplasia with megakaryocytic atypia, while the majority had myeloid hyperplasia and/or erythroid hypoplasia, myeloid dysplasia, and aberrant CD7 expression on blasts. Mutations in growth signaling pathways (RAS or JAK2) were noted at diagnosis or acquired during the disease course in 83% of patients. Two patients progressed upon acquisition of FLT3-TKD (acute myeloid leukemia) or KIT (aggressive systemic mastocytosis) mutations. The prognosis is poor with only two long-term survivors, thus far, who underwent blood or marrow transplantation. We propose that the presence of co-occurring ASXL1, SRSF2, and SETBP1 mutations can be diagnostic of a subtype of MDS/MPN with neutrophilia if clinical and morphological findings align. Our report underscores the association between genotype and phenotype within MDS/MPN and that genomic signatures should guide categorization of these entities., Competing Interests: Declaration of Competing Interest Jain: Institutional research support from CTI Biopharma, Kartos Therapeutics, Incyte; Advisory board participation with Care Dx, Bristol Myers Squibb, Incyte, Abbvie, CTI, Kite, Cogent Biosciences, Blueprint Medicine, Telios pharma, Protagonist therapeutics. Levis: Research funding from Astellas, FujiFilm; Consultancy for Abbvie, Amgen, Bristol-Myers Squibb, Daiichi-Sankyo, Jazz, Menarini, Syndax. The remaining authors report no potential relevant conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Better understanding of the biology of resistance to DNA methyltransferase (DNMT) inhibitors is required to identify therapies that can improve their efficacy for patients with high-risk myelodysplastic syndrome (MDS). CCRL2 is an atypical chemokine receptor that is upregulated in CD34+ cells from MDS patients and induces proliferation of MDS and secondary acute myeloid leukemia (sAML) cells. In this study, we evaluated any role that CCRL2 may have in the regulation of pathways associated with poor response or resistance to DNMT inhibitors. We found that CCRL2 knockdown in TF-1 cells downregulated DNA methylation and PRC2 activity pathways and increased DNMT suppression by azacitidine in MDS/sAML cell lines (MDS92, MDS-L and TF-1). Consistently, CCRL2 deletion increased the sensitivity of these cells to azacitidine in vitro and the efficacy of azacitidine in an MDS-L xenograft model. Furthermore, CCRL2 overexpression in MDS-L and TF-1 cells decreased their sensitivity to azacitidine. Finally, CCRL2 levels were higher in CD34+ cells from MDS and MDS/myeloproliferative neoplasm patients with poor response to DNMT inhibitors. In conclusion, we demonstrated that CCRL2 modulates epigenetic regulatory pathways, particularly DNMT levels, and affects the sensitivity of MDS/sAML cells to azacitidine. These results support CCRL2 targeting as having therapeutic potential in MDS/sAML.

Among the most common genetic alterations in the myelodysplastic syndromes (MDS) are mutations in the spliceosome gene SF3B1 . Such mutations induce specific RNA missplicing events, directly promote ring sideroblast (RS) formation, generally associate with more favorable prognosis, and serve as a predictive biomarker of response to luspatercept. However, not all SF3B1 mutations are the same, and here we report that the E592K variant of SF3B1 associates with high-risk disease features in MDS, including a lack of RS, increased myeloblasts, a distinct co-mutation pattern, and decreased survival. Moreover, in contrast to canonical SF3B1 mutations, E592K induces a unique RNA missplicing pattern, retains an interaction with the splicing factor SUGP1 , and preserves normal RNA splicing of the sideroblastic anemia genes TMEM14C and ABCB7. These data expand our knowledge of the functional diversity of spliceosome mutations, and they suggest that patients with E592K should be approached differently from low-risk, luspatercept-responsive MDS patients with ring sideroblasts and canonical SF3B1 mutations., Competing Interests: Competing Interests The authors declare no competing interests.

The combination of venetoclax and hypomethylating agent (HMA/venetoclax) has emerged as a treatment option for patients with de novo acute myeloid leukemia (AML) who are unfit to receive intensive chemotherapy. In this single-center retrospective study, we evaluated clinical outcomes following treatment with HMA/venetoclax in 35 patients with advanced myeloproliferative neoplasms, myelodysplastic syndrome/myeloproliferative neoplasm overlap syndromes or AML with extramedullary disease. The composite complete remission (CR) rate (including confirmed/presumed complete cytogenetic response, acute leukemia response-complete, CR and CR with incomplete hematologic recovery) was 42.9% with median overall survival (OS) of 9.7 months. Complex karyotype was associated with inferior median OS (3.7 versus 12.2 months; p  = 0.0002) and composite CR rate (22% versus 50.0%; p  = 0.2444). Although SRSF2 mutations were associated with higher composite CR rate (80.0% versus 28.0%; p  = 0.0082), this was not associated with longer median OS (10.9 versus 8.0 months; p  = 0.2269). Future studies should include these patient subgroups.

Patients age ≥55 years with acute lymphoblastic leukemia (ALL) fare poorly with conventional chemotherapy, with a 5-year overall survival (OS) of ∼20%. Tyrosine kinase inhibitors and novel B cell-targeted therapies can improve outcomes, but rates of relapse and death in remission remain high. Allogeneic blood or marrow transplantation (alloBMT) provides an alternative consolidation strategy, and post-transplantation cyclophosphamide (PTCy) facilitates HLA-mismatched transplantations with low rates of nonrelapse mortality (NRM) and graft-versus-host disease (GVHD). The transplantation database at Johns Hopkins was queried for patients age ≥55 years who underwent alloBMT for ALL using PTCy. The database included 77 such patients. Most received reduced-intensity conditioning (RIC) (88.3%), were in first complete remission (CR1) (85.7%), and had B-lineage disease (90.9%). For the entire cohort, 5-year relapse-free survival (RFS) and overall survival (OS) were 46% (95% confidence interval [CI], 34% to 57%) and 49% (95% CI, 37% to 60%), respectively. Grade III-IV acute GVHD occurred in only 3% of patients, and chronic GVHD occurred in 13%. In multivariable analysis, myeloablative conditioning led to worse RFS (hazard ratio [HR], 4.65; P = .001), whereas transplantation in CR1 (HR, .30; P = .004) and transplantation for Philadelphia chromosome-positive (Ph + ) ALL versus T-ALL (HR, .29; P = .03) were associated with improved RFS. Of the 54 patients who underwent RIC alloBMT in CR1 for B-ALL, the 5-year RFS and OS were 62% (95% CI, 47% to 74%) and 65% (95% CI, 51% to 77%), respectively, with a 5-year relapse incidence of 16% (95% CI, 7% to 27%) and an NRM of 24% (95% CI, 13% to 36%). RIC alloBMT with PTCy in CR1 represents a promising consolidation strategy for B-ALL patients age ≥55 years., (Copyright © 2022 The American Society for Transplantation and Cellular Therapy. All rights reserved.)

Precise and reliable cell-specific gene delivery remains technically challenging. Here we report a splicing-based approach for controlling gene expression whereby separate translational reading frames are coupled to the inclusion or exclusion of mutated, frameshifting cell-specific alternative exons. Candidate exons are identified by analyzing thousands of publicly available RNA sequencing datasets and filtering by cell specificity, conservation, and local intron length. This method, which we denote splicing-linked expression design (SLED), can be combined in a Boolean manner with existing techniques such as minipromoters and viral capsids. SLED can use strong constitutive promoters, without sacrificing precision, by decoupling the tradeoff between promoter strength and selectivity. AAV-packaged SLED vectors can selectively deliver fluorescent reporters and calcium indicators to various neuronal subtypes in vivo. We also demonstrate gene therapy utility by creating SLED vectors that can target PRPH2 and SF3B1 mutations. The flexibility of SLED technology enables creative avenues for basic and translational research., (© 2022. The Author(s).)

There are currently no known predictors of myelodysplastic syndrome (MDS)/myeloproliferative overlap neoplasm (MPN) patients' response to hypomethylating agents (HMA). Forty-three patients with MDS/MPN who were treated with HMA during chronic phase and had next-generation sequencing using the established 63-genes panel were identified. Complete and partial remission and marrow response were assessed based on the MDS/MPN International Working Group response criteria. On univariate analysis, younger age, higher number of mutations, and mutations in SETBP1 , RUNX1 , or EZH2 were associated with no response. Multivariable analysis for modeling response were conducted via least absolute shrinkage and selection operator logistic regression approach, and showed that mutations in SETBP1 , RUNX1 , or EZH2 predict lack of HMA response. While limited by sample size, our findings suggest that genomic landscape can potentially identify MDS/MPN patients with lower likelihood of response to HMA.

We describe outcomes after post-transplantation cyclophosphamide and nonmyeloablative conditioning-based allogeneic blood or marrow transplantation for myelofibrosis using matched or mismatched related or unrelated donors. The conditioning regimen consisted of fludarabine, cyclophosphamide, and total body irradiation. Forty-two patients were included, with a median age of 63 years, of whom 19% had Dynamic International Prognostic Scoring System (DIPSS)-plus intermediate-1 risk, 60% had intermediate-2 risk, and 21% had high-risk disease, and 60% had at least 1 high-risk somatic mutation. More than 90% of patients engrafted neutrophils, at a median of 19.5 days, and 7% experienced graft failure. At 1 year and 3 years, respectively, overall survival was 65% and 60%, relapse-free survival was 65% and 31%, relapse was 5% and 40%, and nonrelapse mortality was 30% and 30%. Acute graft-versus-host disease grade 3-4 was seen in 17% of patients at 1 year, and chronic graft-versus-host disease requiring systemic therapy in occurred in 12% patients. Spleen size ≥17 cm or prior splenectomy was associated with inferior relapse-free survival (hazard ratio [HR], 3.50; 95% confidence interval [CI], 1.18 to 10.37; P = .02) and higher relapse rate (subdistribution HR [SDHR] not calculable; P = .01). Age >60 years (SDHR, 0.26; 95% CI, 0.08 to 0.80, P = .02) and receipt of peripheral blood grafts (SDHR, 0.34; 95% CI, 0.11 to 0.99; P = .05) were associated with a lower risk of relapse. In our limited sample, the presence of a high-risk mutation was not statistically significantly associated with an inferior outcome, although ASXL1 was suggestive of inferior survival (SDHR, 2.36; 95% CI, 0.85 to 6.6; P = .09). Overall, this approach shows outcomes comparable those of to previously reported approaches and underscores the importance of spleen size in the evaluation of transplantation candidates., Competing Interests: Declaration of Competing Interest T.J. reports institutional research support from CTI Biopharma and Syneos Health, consultancy for Targeted Healthcare Communications, and advisory board participation for Care Dx, Bristol Myers Squibb, Incyte and CTI Biopharma. The other authors have no conflicts of interest to report., (Copyright © 2022 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Macrophages within the bone marrow (BM) microenvironment take on unexpected roles in acute myeloid leukemia (AML) as reported by Moore and colleagues in this issue of the JCI. In contrast to solid tumors, where tumor-associated macrophages frequently assume an immunosuppressive phenotype that promotes tumor progression, this study revealed that BM macrophages repressed leukemia expansion in AML through a pathway called LC3-associated phagocytosis (LAP). After phagocytosis of dead and dying leukemic cells, including the mitochondria within the leukemic blasts, mitochondrial DNA activated stimulator of IFN genes (STING), leading to inflammatory signals that enhanced phagocytosis and restrained leukemic cell expansion. These findings unveil the modulation of macrophage-mediated phagocytosis via LAP as a potential therapeutic strategy directed at the BM microenvironment in AML.

The identification of new pathways supporting the myelodysplastic syndrome (MDS) primitive cells growth is required to develop targeted therapies. Within myeloid malignancies, men have worse outcomes than women, suggesting male sex hormone-driven effects in malignant hematopoiesis. Androgen receptor promotes the expression of five granulocyte colony-stimulating factor receptor-regulated genes. Among them, CCRL2 encodes an atypical chemokine receptor regulating cytokine signaling in granulocytes, but its role in myeloid malignancies is unknown. Our study revealed that CCRL2 is up-regulated in primitive cells from patients with MDS and secondary acute myeloid leukemia (sAML). CCRL2 knockdown suppressed MDS92 and MDS-L cell growth and clonogenicity in vitro and in vivo and decreased JAK2/STAT3/STAT5 phosphorylation. CCRL2 coprecipitated with JAK2 and potentiated JAK2-STAT interaction. Erythroleukemia cells expressing JAK2V617F showed less effect of CCRL2 knockdown, whereas fedratinib potentiated the CCRL2 knockdown effect. Conclusively, our results implicate CCRL2 as an MDS/sAML cell growth mediator, partially through JAK2/STAT signaling.

Activating variants in the PEST region of NOTCH1 have been associated with aggressive phenotypes in human cancers, including triple-negative breast cancer (TNBC). Previous studies suggested that PEST domain variants in TNBC patients resulted in increased cell proliferation, invasiveness, and decreased overall survival. In this study, we assess the phenotypic transformation of activating NOTCH1 variants and their response to standard of care therapies. AAV-mediated gene targeting was used to isogenically incorporate 3 NOTCH1 variants, including a novel TNBC frameshift variant, in two non-tumorigenic breast epithelial cell lines, MCF10A and hTERT-IMEC. Two different variants at the NOTCH1 A2241 site (A2441fs and A2441T) both demonstrated increased transformative properties when compared to a non-transformative PEST domain variant (S2523L). These phenotypic changes include proliferation, migration, anchorage-independent growth, and MAPK pathway activation. In contrast to previous studies, activating NOTCH1 variants did not display sensitivity to a gamma secretase inhibitor (GSI) or resistance to chemotherapies. This study demonstrates distinct transformative phenotypes are specific to a given variant within NOTCH1 and these phenotypes do not correlate with sensitivities or resistance to chemotherapies or GSIs. Although previous studies have suggested NOTCH1 variants may be prognostic for TNBC, our study does not demonstrate prognostic ability of these variants and suggests further characterization would be required for clinical applications., Competing Interests: CONFLICTS OF INTEREST B.H.P. is a paid consultant for Jackson Labs, EQRx, Sermonix, Hologics, Guardant Health and is a paid scientific advisory board member for Celcuity Inc. B.H.P. also has research contracts with GE Healthcare, Lilly and Pfizer. Under separate licensing agreements between Horizon Discovery, LTD and The Johns Hopkins University, B.H.P. is entitled to a share of royalties received by the University on sales of products. The terms of this arrangement are being managed by the Johns Hopkins University in accordance with its conflict of interest policies. J.D. receives royalties from patents held by City of Hope and Thomas Jefferson University. J.D. also holds stock awards from Xilio Therapeutics. None of these listed had do direct conflict of interest on the project., (Copyright: © 2022 Cravero et al.)

Introduction: Risk stratification is crucial to the appropriate management of many diseases, but in patients with myelodysplastic syndromes (MDS), for whom expected survival can vary greatly, accurate disease prognostication is especially important. This is further supported by a relative lack of therapies in MDS, and thus we must prognosticate carefully and accurately. Currently, patients with MDS are often grouped into higher-risk (HR) versus lower-risk (LR) disease using clinical prognostic scoring systems, but these systems have limitations., Areas Covered: The authors reviewed the literature on diagnostics, prognostics, therapeutics and outcomes in MDS. Factors such as disease etiology, specific clinical characteristics, or molecular genetic information not captured in the international prognostic scoring system revised IPSS-R can alter risk stratification, and identify a subset of LR-MDS patients who actually behave more like HR-MDS., Expert Opinion: This review will describe the current identification and management of patients with LR MDS disease whose condition is likely to behave in a less favorable manner than predicted by the IPSS-R. The authors comment on clinical and molecular features which are believe to upstage a patient from lower to higher risk disease.

Allogeneic blood or marrow transplantation (alloBMT) is standard of care for adults with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) in first complete remission (CR1). The routine pretransplant and posttransplant use of tyrosine kinase inhibitors (TKIs) has dramatically improved outcomes, but the optimal conditioning regimen, donor type, and TKI remain undefined. The bone marrow transplant database at Johns Hopkins was queried for adult patients with de novo Ph+ ALL who received alloBMT using posttransplantation cyclophosphamide (PTCy) as a component of graft-versus-host disease (GVHD) prophylaxis from 2008 to 2018. Among transplants for Ph+ ALL, 69 (85%) were performed in CR1, and 12 (15%) were performed in second or greater remission (CR2+). The majority of transplants (58%) were HLA haploidentical. Nearly all patients (91.4%) initiated TKI posttransplant. For patients in CR1, the 5-year relapse-free survival (RFS) was 66%. The use of nonmyeloablative conditioning, absence of measurable residual disease (MRD) according to flow cytometry at transplant, and the use of dasatinib vs imatinib at diagnosis were associated with improved overall survival (OS) and RFS. Neither donor type nor recipient age ≥60 years affected RFS. When analyzing all transplants, alloBMT in CR1 (vs CR2+) and the absence of pretransplant MRD were associated with improved RFS. Most relapses were associated with the emergence of kinase domain mutations. The cumulative incidence of grade 3 to 4 acute GVHD at 1 year was 9%, and moderate to severe chronic GVHD at 2 years was 8%. Nonmyeloablative alloBMT with PTCy for Ph+ ALL in an MRD-negative CR1 after initial treatment with dasatinib yields favorable outcomes., (© 2020 by The American Society of Hematology.)

Phosphoglycerate dehydrogenase (PHGDH) catalyzes the first step in the synthesis of the amino acid serine, important for protein synthesis, one-carbon metabolism, lipid production, redox homeostasis, and other key processes of normal and cancer metabolism. While PHGDH is often overexpressed in cancer cells, how it is regulated has been unclear. In this issue of the JCI, Liu and colleagues describe a new aspect of PHGDH regulation, demonstrating that the Parkinson disease gene and tumor suppressor Parkin bound and ubiquitinated PHGDH. Parkin promoted PHGDH degradation, suppressed serine synthesis, and inhibited tumor growth in human cancer cell line xenografts. Conversely, inactivation of Parkin not only accelerated tumor growth, but also sensitized tumors to small molecule inhibitors of PHGDH. These results offer a new link between Parkin and the serine synthesis pathway, and they bear translational potential that warrants further study in Parkin-deficient human cancers.

Mutations in the splicing factor 3b subunit 1 ( SF3B1 ) gene create a neomorphic protein that disrupts RNA splicing, but the downstream consequences of this missplicing are unclear. Our recent study of isogenic human cells demonstrated that SF3B1 MUT induces reprogramming of energy metabolism and a targetable vulnerability to deprivation of the nonessential amino acid serine., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Cancer-associated mutations in the spliceosome gene SF3B1 create a neomorphic protein that produces aberrant mRNA splicing in hundreds of genes, but the ensuing biologic and therapeutic consequences of this missplicing are not well understood. Here we have provided evidence that aberrant splicing by mutant SF3B1 altered the transcriptome, proteome, and metabolome of human cells, leading to missplicing-associated downregulation of metabolic genes, decreased mitochondrial respiration, and suppression of the serine synthesis pathway. We also found that mutant SF3B1 induces vulnerability to deprivation of the nonessential amino acid serine, which was mediated by missplicing-associated downregulation of the serine synthesis pathway enzyme PHGDH. This vulnerability was manifest both in vitro and in vivo, as dietary restriction of serine and glycine in mice was able to inhibit the growth of SF3B1MUT xenografts. These findings describe a role for SF3B1 mutations in altered energy metabolism, and they offer a new therapeutic strategy against SF3B1MUT cancers.

Purpose: Estrogen receptor-alpha (ER) is a therapeutic target of ER-positive (ER+) breast cancers. Although ER signaling is complex, many mediators of this pathway have been identified. Specifically, phosphorylation of ER at serine 118 affects responses to estrogen and therapeutic ligands and has been correlated with clinical outcomes in ER+ breast cancer patients. We hypothesized that a newly described germline variant (S118P) at this residue would drive cellular changes consistent with breast cancer development and/or hormone resistance., Methods: Isogenic human breast epithelial cell line models harboring ER S118P were developed via genome editing and characterized to determine the functional effects of this variant. We also examined the frequency of ER S118P in a case-control study (N = 536) of women with and without breast cancer with a familial risk., Results: In heterozygous knock-in models, the S118P variant demonstrated no significant change in proliferation, migration, MAP Kinase pathway signaling, or response to the endocrine therapies tamoxifen and fulvestrant. Further, there was no difference in the prevalence of S118P between women with and without cancer relative to population registry databases., Conclusions: This study suggests that the ER S118P variant does not affect risk for breast cancer or hormone therapy resistance. Germline screening and modification of treatments for patients harboring this variant are likely not warranted.

Background: Molecular-based diagnostics have great utility for cancer detection. We have used droplet digital PCR (ddPCR) as a platform for identifying mutations in circulating plasma tumor DNA (ptDNA). We present the unexpected finding of a spurious mutant allele fraction that was discovered to be artifactual because of the presence of a single-nucleotide polymorphism (SNP) in a patient sample., Design and Methods: Probe and primer combinations for the K700 and V701 loci of the SF3B1 spliceosome gene were designed for ddPCR to identify the percentage of mutant and wild-type alleles. Clinical samples from patients with cancer with known SF3B1 mutations were collected and tested to evaluate the assays' ability to detect SF3B1 mutations in ptDNA., Results: Patient samples showed SF3B1 K700E mutations within the ptDNA of 4 patients with acute leukemia and 3 with myelodysplastic syndrome who were known to harbor this mutation. A blood sample from a patient with lung cancer with a known SF3B1 V701F mutation was also analyzed and this mutation was successfully identified in ptDNA. However, 1 of the patients with a K700E mutation was found to have a mutational burden of 98%. After careful analysis of this locus by Sanger sequencing and ddPCR, this patient was found to have an SNP (R702R), which prevented binding of the ddPCR wild-type probe to its cognate allele., Conclusions: These results further support that ddPCR-based assays may be valuable companion diagnostics for the identification and monitoring of patients with cancer, but the results also emphasize the need to identify SNPs at loci that are being analyzed., (© 2017 American Association for Clinical Chemistry.)

Background/purpose: The combined contributions of oncogenes and tumor suppressor genes toward carcinogenesis remain poorly understood. Elucidation of cancer gene cooperativity can provide new insights leading to more effective use of therapies., Experimental Design/methods: We used somatic cell genome editing to introduce singly and in combination PIK3CA mutations (E545K or H1047R) with TP53 alterations (R248W or knockout), to assess any enhanced cancerous phenotypes. The non-tumorigenic human breast epithelial cell line, MCF10A, was used as the parental cell line, and resultant cells were assessed via various in vitro assays, growth as xenografts, and drug sensitivity assays using targeted agents and chemotherapies., Results: Compared to single-gene-targeted cells and parental controls, cells with both a PIK3CA mutation and TP53 alteration had increased cancerous phenotypes including cell proliferation, soft agar colony formation, aberrant morphology in acinar formation assays, and genomic heterogeneity. Cells also displayed varying sensitivities to anti-neoplastic drugs, although all cells with PIK3CA mutations showed a relative increased sensitivity to paclitaxel. All cell lines remained non-tumorigenic., Conclusions: This cell line panel provides a resource for further elucidating cooperative genetic mediators of carcinogenesis and response to therapies.

Purpose: Tumor genomic profiling for personalized oncology therapy is being widely applied in clinical practice even as it is being evaluated more formally in clinical trials. Given the complexities of genomic data and its application to clinical use, molecular tumor boards with diverse expertise can provide guidance to oncologists and patients seeking to implement personalized genetically targeted therapy in practice., Methods: A multidisciplinary molecular tumor board reviewed tumor molecular profiling reports from consecutive referrals at the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins over a 3-year period. The tumor board weighed evidence for actionability of genomic alterations identified by molecular profiling and provided recommendations including US Food and Drug Administration-approved drug therapy, clinical trials of matched targeted therapy, off-label use of such therapy, and additional tumor or germline genetic testing., Results: One hundred fifty-five patients were reviewed. Actionable genomic alterations were identified in 132 patients (85%). Off-label therapies were recommended in 37 patients (24%). Eleven patients were treated off-label, and 13 patients were enrolled onto clinical trials of matched targeted therapies. Median progression-free survival of patients treated with matched therapies was 5 months (95% CI, 2.9 months to not reached), and the progression-free survival probability at 6 months was 43%(95% CI, 26% to 71%). Lack of locally available clinical trials was the major limitation on clinical actionability of tumor profiling reports., Conclusion: The molecular tumor board recommended off-label targeted therapies for a quarter of all patients reviewed. Outcomes were heterogeneous, although 43% of patients receiving genomically matched therapy derived clinical benefit lasting at least 6 months. Until more data become available from precision oncology trials, molecular tumor boards can help guide appropriate use of tumor molecular testing to direct therapy.

Next-generation sequencing (NGS) is increasingly being used in cancer care to identify both somatic tumor driver mutations that can be targeted for therapy, and heritable mutations in the germline associated with increased cancer risk. This report presents a case of a JAK2 V617F mutation falsely identified as a duodenal cancer mutation via NGS. The patient was found to have a history of polycythemia vera, a disorder with a high incidence of JAK2 somatic mutations. Buccal cell DNA showed heterozygosity for the mutation, suggesting that it was potentially germline. However, subsequent resequencing of tumor, adjacent normal tissue, and fingernail DNA confirmed the mutation was somatic, and its presence in tumor and buccal cells resulted from contaminating blood cells. This report highlights important nuances of NGS that can lead to misinterpretation of results with potential clinical implications., (Copyright © 2016 by the National Comprehensive Cancer Network.)

Purpose: Mutations in the estrogen receptor (ER)α gene, ESR1, have been identified in breast cancer metastases after progression on endocrine therapies. Because of limitations of metastatic biopsies, the reported frequency of ESR1 mutations may be underestimated. Here, we show a high frequency of ESR1 mutations using circulating plasma tumor DNA (ptDNA) from patients with metastatic breast cancer., Experimental Design: We retrospectively obtained plasma samples from eight patients with known ESR1 mutations and three patients with wild-type ESR1 identified by next-generation sequencing (NGS) of biopsied metastatic tissues. Three common ESR1 mutations were queried for using droplet digital PCR (ddPCR). In a prospective cohort, metastatic tissue and plasma were collected contemporaneously from eight ER-positive and four ER-negative patients. Tissue biopsies were sequenced by NGS, and ptDNA ESR1 mutations were analyzed by ddPCR., Results: In the retrospective cohort, all corresponding mutations were detected in ptDNA, with two patients harboring additional ESR1 mutations not present in their metastatic tissues. In the prospective cohort, three ER-positive patients did not have adequate tissue for NGS, and no ESR1 mutations were identified in tissue biopsies from the other nine patients. In contrast, ddPCR detected seven ptDNA ESR1 mutations in 6 of 12 patients (50%)., Conclusions: We show that ESR1 mutations can occur at a high frequency and suggest that blood can be used to identify additional mutations not found by sequencing of a single metastatic lesion., Competing Interests: of Potential Conflicts of Interest: B.H.P. is a paid consultant for Novartis and is a member of the scientific advisory boards of Horizon Discovery, LTD and Loxo Oncology, and has research contracts with Genomic Health, Inc and Foundation Medicine. Under separate licensing agreements between Horizon Discovery, LTD and The Johns Hopkins University, B.H.P. is entitled to a share of royalties received by the University on sales of products. The terms of this arrangement are being managed by the Johns Hopkins University, in accordance with its conflict of interest policies. D.F.H. has received clinical and laboratory research funding from Janssen, AstraZeneca, and Pfizer. The University of Michigan holds two patents regarding isolation and characterization of circulating tumor cells, one of which has been licensed to Janssen, naming D.F.H. as the inventor. D.F.H. also has stock options in two diagnostic companies: InBiomotion and OncImmune. None of these conflicts pertains to this manuscript. All other authors declare no potential conflicts., (©2015 American Association for Cancer Research.)

Recurrent human epidermal growth factor receptor 2 (HER2) missense mutations have been reported in human cancers. These mutations occur primarily in the absence of HER2 gene amplification such that most HER2-mutant tumors are classified as "negative" by FISH or immunohistochemistry assays. It remains unclear whether nonamplified HER2 missense mutations are oncogenic and whether they are targets for HER2-directed therapies that are currently approved for the treatment of HER2 gene-amplified breast cancers. Here we functionally characterize HER2 kinase and extracellular domain mutations through gene editing of the endogenous loci in HER2 nonamplified human breast epithelial cells. In in vitro and in vivo assays, the majority of HER2 missense mutations do not impart detectable oncogenic changes. However, the HER2 V777L mutation increased biochemical pathway activation and, in the context of a PIK3CA mutation, enhanced migratory features in vitro. However, the V777L mutation did not alter in vivo tumorigenicity or sensitivity to HER2-directed therapies in proliferation assays. Our results suggest the oncogenicity and potential targeting of HER2 missense mutations should be considered in the context of cooperating genetic alterations and provide previously unidentified insights into functional analysis of HER2 mutations and strategies to target them.

The tumor protein 53 (TP53) tumor suppressor gene is the most frequently somatically altered gene in human cancers. Here we show expression of N-Myc down-regulated gene 1 (NDRG1) is induced by p53 during physiologic low proliferative states, and mediates centrosome homeostasis, thus maintaining genome stability. When placed in physiologic low-proliferating conditions, human TP53 null cells fail to increase expression of NDRG1 compared with isogenic wild-type controls and TP53 R248W knockin cells. Overexpression and RNA interference studies demonstrate that NDRG1 regulates centrosome number and amplification. Mechanistically, NDRG1 physically associates with γ-tubulin, a key component of the centrosome, with reduced association in p53 null cells. Strikingly, TP53 homozygous loss was mutually exclusive of NDRG1 overexpression in over 96% of human cancers, supporting the broad applicability of these results. Our study elucidates a mechanism of how TP53 loss leads to abnormal centrosome numbers and genomic instability mediated by NDRG1.

Cells engage sophisticated programs of DNA damage response (DDR) and repair to guard against genetic mutations. Although there is significant knowledge concerning DDR in interphase cells, much less is known about these processes in mitosis. Direct interaction between MDC1, a master DDR organizer, and a marker of DNA damage, histone γH2AX, is required to trigger robust repair. Here we show that the DNA damage-induced interaction between MDC1 and γH2AX is attenuated in mitosis. Furthermore, inhibition in the activity of the core mitotic regulator CDK1, either by pharmacologic inhibition or siRNA attenuation, enhances MDC1-γH2AX colocalization in mitosis. Our findings offer key new insights into how DDR is controlled during mitosis., (©2012 AACR.)

We have previously shown that a non-toxic noscapinoid, EM011 binds tubulin without altering its monomer/polymer ratio. EM011 is more active than the parent molecule, noscapine, in inducing G2/M arrest, inhibiting cellular proliferation and tumor growth in various human xenograft models. However, the mechanisms of mitotic-block and subsequent cell death have remained elusive. Here, we show that EM011-induced attenuation of microtubule dynamics was associated with impaired association of microtubule plus-end tracking proteins, such as EB1 and CLIP-170. EM011 treatment then led to the formation of multipolar spindles containing 'real' centrioles indicating drug-induced centrosome amplification and persistent centrosome declustering. Centrosome amplification was accompanied by an upregulation of Aurora A and Plk4 protein levels, as well as a surge in the kinase activity of Aurora A, suggesting a deregulation of the centrosome duplication cycle. Cell-cycle phase-specific experiments showed that the 'cytotoxicity-window' of the drug encompasses the late S-G2 period. Drug-treatment, excluding S-phase, not only resulted in lower sub-G1 population but also attenuated centrosome amplification and spindle multipolarity, suggesting that drug-induced centrosome amplification is essential for maximal cell death. Subsequent to a robust mitotic arrest, EM011-treated cells displayed diverse cellular fates suggesting a high degree of intraline variation. Some 'apoptosis-evasive' cells underwent aberrant cytokinesis to generate rampant aneuploidy that perhaps contributed to drug-induced cell death. These data indicate that spindle multipolarity induction by means of centrosome amplification has an exciting chemotherapeutic potential that merits further investigation., (© 2011 Macmillan Publishers Limited)

The p53 tumor suppressor inhibits the proliferation of cells that undergo prolonged activation of the mitotic checkpoint. However, the function of this antiproliferative response is not well defined. Here, we report that p53 suppresses structural chromosome instability after mitotic arrest in human cells. In both HCT116 colon cancer cells and normal human fibroblasts, DNA breaks occurred during mitotic arrest in a p53-independent manner, but p53 was required to suppress the proliferation and structural chromosome instability of the resulting polyploid cells. In contrast, cells made polyploid without mitotic arrest exhibited neither significant structural chromosome instability nor p53-dependent cell cycle arrest. We also observed that p53 suppressed both the frequency and structural chromosome instability of spontaneous polyploids in HCT116 cells. Furthermore, time-lapse videomicroscopy revealed that polyploidization of p53(-/-) HCT116 cells is frequently accompanied by mitotic arrest. These data suggest that a function of the p53-dependent postmitotic response is the prevention of structural chromosome instability after prolonged activation of the mitotic checkpoint. Accordingly, our study suggests a novel mechanism of tumor suppression for p53, as well as a potential function for p53 in the outcome of antimitotic chemotherapy.

Mitotic abnormalities are a common feature of human cancer cells, and recent studies have provided evidence that such abnormalities may play a causative, rather than merely incidental role, in tumorigenesis. One such abnormality is prolonged activation of the mitotic checkpoint, which can be provoked by a number of the gene changes that drive tumor formation. At the same time, antimitotic chemotherapeutics exert their clinical efficacy through the large-scale induction of prolonged mitotic checkpoint activation, indicating that mitotic arrest is influential in both the formation and treatment of human cancer. However, how this influence occurs is not well understood. In this perspective, we will discuss the current evidence in support of the potential mechanisms by which prolonged activation of the mitotic checkpoint affects both tumorigenesis and antimitotic chemotherapy.

Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with tumor suppressive activity in colorectal cancer. Here, we investigated whether KLF4 is involved in maintaining genetic stability in mouse embryonic fibroblasts (MEFs) isolated from mice wild type (+/+), heterozygous (+/-), or homozygous (-/-) for the Klf4 alleles. Compared to Klf4(+/+) and Klf4(+/-) MEFs, Klf4(-/-) MEFs had both a higher level of apoptosis and rate of proliferation. Quantification of chromosome numbers showed that Klf4(-/-) MEFs were aneuploid. A higher number of Klf4(-/-) MEFs exhibited gamma-H2AX foci and had higher amounts of gamma-H2AX compared to controls. Cytogenetic analysis demonstrated the presence of numerous chromosome aberrations including dicentric chromosomes, chromatid breaks, and double minute chromosomes in Klf4(-/-) cells but in few, if any, Klf4(+/+) or Klf4(+/-) MEFs. Approximately 25% of Klf4(-/-) MEFs exhibited centrosome amplification in contrast to the less than 5% of Klf4(+/+) or Klf4(+/-) MEFs. Finally, only Klf4(-/-) MEFs were capable of anchorage-independent growth. Taken together, these findings demonstrate that MEFs null for the Klf4 alleles are genetically unstable, as evidenced by the presence of aneuploidy, chromosome aberration and centrosome amplification. The results support a crucial role for KLF4 in maintaining genetic stability and as a tumor suppressor.

The mitotic checkpoint is a mechanism that arrests the progression to anaphase until all chromosomes have achieved proper attachment to mitotic spindles. In cancer cells, satisfaction of this checkpoint is frequently delayed or prevented by various defects, some of which have been causally implicated in tumorigenesis. At the same time, deliberate induction of mitotic arrest has proved clinically useful, as antimitotic drugs that interfere with proper chromosome-spindle interactions are effective anticancer agents. However, how mitotic arrest contributes to tumorigenesis or antimitotic drug toxicity is not well defined. Here, we report that mitotic chromosomes can acquire DNA breaks during both pharmacologic and genetic induction of mitotic arrest in human cancer cells. These breaks activate a DNA damage response, occur independently of cell death, and subsequently manifest as karyotype alterations. Such breaks can also occur spontaneously, particularly in cancer cells containing mitotic spindle abnormalities. Moreover, we observed evidence of some breakage in primary human cells. Our findings thus describe a novel source of DNA damage in human cells. They also suggest that mitotic arrest may promote tumorigenesis and antimitotic toxicity by provoking DNA damage.

Mitosis is a crucial part of the cell cycle. A successful mitosis requires the proper execution of many complex cellular behaviors. Thus, there are many points at which mitosis may be disrupted. In cancer cells, chronic disruption of mitosis can lead to unequal segregation of chromosomes, a phenomenon known as chromosomal instability. A majority of colorectal tumors suffer from this instability, and recent studies have begun to reveal the specific ways in which mitotic defects promote chromosomal instability in colorectal cancer.

Prostate cancer is the most commonly diagnosed noncutaneous neoplasm and second most common cause of cancer-related mortality in western men. To investigate the mechanisms of prostate cancer development and progression, we did expression profiling of human prostate cancer and benign tissues. We show that the SOX4 is overexpressed in prostate tumor samples compared with benign tissues by microarray analysis, real-time PCR, and immunohistochemistry. We also show that SOX4 expression is highly correlated with Gleason score at the mRNA and protein level using tissue microarrays. Genes affected by SOX4 expression were also identified, including BCL10, CSF1, and NcoA4/ARA70. TLE-1 and BBC3/PUMA were identified as direct targets of SOX4. Silencing of SOX4 by small interfering RNA transfection induced apoptosis of prostate cancer cells, suggesting that SOX4 could be a therapeutic target for prostate cancer. Stable transfection of SOX4 into nontransformed prostate cells enabled colony formation in soft agar, suggesting that, in the proper cellular context, SOX4 can be a transforming oncogene.

We previously showed that the zinc finger-containing transcription factor Krüppel-like factor 5 (KLF5) is important in mediating transformation by oncogenic H-Ras through induction of cyclin D1 expression and acceleration of the G1/S transition of the cell cycle. Here we present evidence of a role for KLF5 in accelerating mitotic entry in H-Ras-transformed NIH3T3 fibroblasts. When compared with non-transformed parental NIH3T3 cells, H-Ras-transformed fibroblasts exhibit an increase in mitotic index, levels of cyclin B1 and Cdc2, and cyclin B1/Cdc2 kinase activity. Inhibition of KLF5 expression in H-Ras-transformed cells with KLF5-specific small interfering RNA (siRNA) results in a decrease in each of the aforementioned parameters, with a concomitant reduction in the transforming potential of the cells. Conversely, over-expression of KLF5 in NIH3T3 cells leads to an increase in the promoter activity of the genes encoding cyclin B1 and Cdc2. These results indicate that KLF5 accelerates mitotic entry in H-Ras-transformed cells by transcriptionally activating cyclin B1 and Cdc2, which leads to an increase in cyclin B1/Cdc2 kinase activity. Extending our previous observation that KLF5 activates cyclin D1 transcription to promote G1/S transition, our current results further support a crucial function for KLF5 in mediating cellular transformation caused by oncogenic H-Ras.

Centrosome duplication is a carefully controlled process in the cell cycle. Previous studies indicate that the tumor suppressor, p53, regulates centrosome duplication. Here, we present evidence for the involvement of the mammalian Krüppel-like transcription factor, KLF4, in preventing centrosome amplification following DNA damage caused by gamma-irradiation. The colon cancer cell line HCT116, which contains wild-type p53 alleles (HCT116 p53+/+), displayed stable centrosome numbers following gamma-irradiation. In contrast, HCT116 cells null for the p53 alleles (HCT116 p53-/-) exhibited centrosome amplification after irradiation. In the latter cell line, KLF4 was not activated following gamma-irradiation due to the absence of p53. However, centrosome amplification could be suppressed in irradiated HCT116 p53-/- cells by conditional induction of exogenous KLF4. Conversely, in a HCT116 p53+/+ cell line stably transfected with small hairpin RNA (shRNA) designed to specifically inhibit KLF4, gamma-irradiation induced centrosome amplification. In these cells, the inability of KLF4 to become activated in response to DNA damage was directly associated with an increase in cyclin E level and Cdk2 activity, both essential for regulating centrosome duplication. Cotransfection experiments showed that KLF4 overexpression suppressed the promoter activity of the cyclin E gene. The results of this study demonstrated that KLF4 is both necessary and sufficient in preventing centrosome amplification following gamma-radiation-induced DNA damage and does so by transcriptionally suppressing cyclin E expression.

Krüppel-like factors (KLFs) are evolutionarily conserved zinc finger-containing transcription factors with diverse regulatory functions in cell growth, proliferation, differentiation, and embryogenesis. KLF4 and KLF5 are two closely related members of the KLF family that have a similar tissue distribution in embryos and adults. However, the two KLFs often exhibit opposite effects on regulation of gene transcription, despite binding to similar, if not identical, cis-acting DNA sequences. In addition, KLF4 and 5 exert contrasting effects on cell proliferation in many instances; while KLF4 is an inhibitor of cell growth, KLF5 stimulates proliferation. Here we review the biological properties and biochemical mechanisms of action of the two KLFs in the context of growth regulation.