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Supplementary Data Legend, Supplementary Table (S7) and Supplementary Figures (S1-S6)

We evaluated clinical significance of recently identified subtypes of acute lymphoblastic leukemia (ALL) in 598 children treated with minimal residual disease (MRD)–directed therapy. Among the 16 B-cell ALL (B-ALL) and 8 T-cell ALL subtypes identified by next-generation sequencing, ETV6–RUNX1, high-hyperdiploid, and DUX4-rearranged B-ALL had the best 5-year event-free survival rates (95.0%–98.4%); TCF3–PBX1, PAX5-altered (PAX5alt), T-cell, early T-cell precursor (ETP), intrachromosomal amplification of chromosome 21 (iAMP21), and hypodiploid ALL intermediate rates (80.0%–88.2%); and BCR–ABL1, BCR–ABL1-like, ETV6–RUNX1-like, and KMT2A-rearranged ALL the worst rates (64.1%–76.2%). All but 3 of the 142 patients with day 8 blood MRD DUX4-rearranged, BCR–ABL1-like, and ZNF384-rearranged ALL, and achievement of day 42 MRD MEF2D-rearranged, and ETV6–RUNX1-like ALL. Thus, new subtypes including DUX4-rearranged, PAX5alt, BCR–ABL1-like, ETV6–RUNX1-like, MEF2D-rearranged, and ZNF384-rearranged ALL have important prognostic and therapeutic implications.Significance:Genomic analyses and MRD should be used together for risk-directed treatment of childhood ALL. Six recently described subtypes—DUX4-rearranged, PAX5alt, BCR–ABL1-like, ETV6–RUNX1-like, MEF2D-rearranged, and ZNF384-rearranged ALL—had prognostic and therapeutic significance with contemporary risk-directed treatment.See related commentary by Segers and Cools, p. 294.See related video from the AACR Annual Meeting 2021: https://vimeo.com/558556916

Supplementary Table S1-S5

Supplementary Table S6

Supplementary Figure S4

Genomic studies of pediatric cancer have primarily focused on specific tumor types or high-risk disease. Here, we used a three-platform sequencing approach, including whole-genome sequencing (WGS), whole-exome sequencing (WES), and RNA sequencing (RNA-seq), to examine tumor and germline genomes from 309 prospectively identified children with newly diagnosed (85%) or relapsed/refractory (15%) cancers, unselected for tumor type. Eighty-six percent of patients harbored diagnostic (53%), prognostic (57%), therapeutically relevant (25%), and/or cancer-predisposing (18%) variants. Inclusion of WGS enabled detection of activating gene fusions and enhancer hijacks (36% and 8% of tumors, respectively), small intragenic deletions (15% of tumors), and mutational signatures revealing of pathogenic variant effects. Evaluation of paired tumor–normal data revealed relevance to tumor development for 55% of pathogenic germline variants. This study demonstrates the power of a three-platform approach that incorporates WGS to interrogate and interpret the full range of genomic variants across newly diagnosed as well as relapsed/refractory pediatric cancers.Significance:Pediatric cancers are driven by diverse genomic lesions, and sequencing has proven useful in evaluating high-risk and relapsed/refractory cases. We show that combined WGS, WES, and RNA-seq of tumor and paired normal tissues enables identification and characterization of genetic drivers across the full spectrum of pediatric cancers.This article is highlighted in the In This Issue feature, p. 2945

PDF - 76K, Legends for Supplementary Figures 1-9.

Involves Supplementary Table Legends and Supplementary Figures and associated legends

Effective data sharing is key to accelerating research to improve diagnostic precision, treatment efficacy, and long-term survival in pediatric cancer and other childhood catastrophic diseases. We present St. Jude Cloud (https://www.stjude.cloud), a cloud-based data-sharing ecosystem for accessing, analyzing, and visualizing genomic data from >10,000 pediatric patients with cancer and long-term survivors, and >800 pediatric sickle cell patients. Harmonized genomic data totaling 1.25 petabytes are freely available, including 12,104 whole genomes, 7,697 whole exomes, and 2,202 transcriptomes. The resource is expanding rapidly, with regular data uploads from St. Jude's prospective clinical genomics programs. Three interconnected apps within the ecosystem—Genomics Platform, Pediatric Cancer Knowledgebase, and Visualization Community—enable simultaneously performing advanced data analysis in the cloud and enhancing the Pediatric Cancer knowledgebase. We demonstrate the value of the ecosystem through use cases that classify 135 pediatric cancer subtypes by gene expression profiling and map mutational signatures across 35 pediatric cancer subtypes.Significance:To advance research and treatment of pediatric cancer, we developed St. Jude Cloud, a data-sharing ecosystem for accessing >1.2 petabytes of raw genomic data from >10,000 pediatric patients and survivors, innovative analysis workflows, integrative multiomics visualizations, and a knowledgebase of published data contributed by the global pediatric cancer community.This article is highlighted in the In This Issue feature, p. 995

PDF - 308K, Supplementary Figure 9. Inhibition of Akt and Erk phosphorylation in vivo by combination treatment.

PDF - 299K, Supplementary Figure 1. HER3 activation is not increased in LapR cells. Supplementary Figure 2. Time-independent maintenance of phospho-Akt during lapatinib treatment in BT474 LapR cells. Supplementary Figure 3. p110α knockdown sensitizes lapatinib-resistant cells to lapatinib. Supplementary Figure 4. p110α knockdown is more effective in BT474 LapR cells than UACC893 LapR cells. Supplementary Figure 5. Wild-type and mutant p110α promote basal proliferation. BT474 cells stably infected with indicated p110α constructs were grown for 4 days. Supplementary Figure 6. Analysis of combination treatment synergy. Supplementary Figure 7. Lapatinib plus BYL719 inhibits HER3. Supplementary Figure 8. Lapatinib plus BYL719 does not induce weight loss.

To investigate the genomic evolution of metastatic pediatric osteosarcoma, we performed whole-genome and targeted deep sequencing on 14 osteosarcoma metastases and two primary tumors from four patients (two to eight samples per patient). All four patients harbored ancestral (truncal) somatic variants resulting in TP53 inactivation and cell-cycle aberrations, followed by divergence into relapse-specific lineages exhibiting a cisplatin-induced mutation signature. In three of the four patients, the cisplatin signature accounted for >40% of mutations detected in the metastatic samples. Mutations potentially acquired during cisplatin treatment included NF1 missense mutations of uncertain significance in two patients and a KIT G565R activating mutation in one patient. Three of four patients demonstrated widespread ploidy differences between samples from the sample patient. Single-cell seeding of metastasis was detected in most metastatic samples. Cross-seeding between metastatic sites was observed in one patient, whereas in another patient a minor clone from the primary tumor seeded both metastases analyzed. These results reveal extensive clonal heterogeneity in metastatic osteosarcoma, much of which is likely cisplatin-induced.Implications:The extent and consequences of chemotherapy-induced damage in pediatric cancers is unknown. We found that cisplatin treatment can potentially double the mutational burden in osteosarcoma, which has implications for optimizing therapy for recurrent, chemotherapy-resistant disease.

Osteosarcoma clinical history and capture validation sequencing data. Supplementary Table S1. Sample and treatment information for relapsed osteosarcoma patients. Supplementary Table S2. SJOS0011101 SNVs detected by capture validation and tumor purity. Supplementary Table S3. SJOS0011105 SNVs detected by capture validation and tumor purity. Supplementary Table S4. SJOS0011107 SNVs detected by capture validation and tumor purity. Supplementary Table S5. SJOS010 SNVs detected by capture validation and tumor purity.

Saracatinib inhibits proliferation and c-Myc, and GLUT1 in MFPs of ER- MMTV-neu* mouse model.

Preventing breast cancer will require the development of targeted strategies that can effectively block disease progression. Tamoxifen and aromatase inhibitors are effective in addressing estrogen receptor–positive (ER+) breast cancer development, but estrogen receptor–negative (ER−) breast cancer remains an unmet challenge due to gaps in pathobiologic understanding. In this study, we used reverse-phase protein array to identify activation of Src kinase as an early signaling alteration in premalignant breast lesions of women who did not respond to tamoxifen, a widely used ER antagonist for hormonal therapy of breast cancer. Src kinase blockade with the small-molecule inhibitor saracatinib prevented the disorganized three-dimensional growth of ER− mammary epithelial cells in vitro and delayed the development of premalignant lesions and tumors in vivo in mouse models developing HER2+ and ER− mammary tumors, extending tumor-free and overall survival. Mechanistic investigations revealed that Src blockade reduced glucose metabolism as a result of an inhibition in ERK1/2–MNK1–eIF4E–mediated cap-dependent translation of c-Myc and transcription of the glucose transporter GLUT1, thereby limiting energy available for cell growth. Taken together, our results provide a sound rationale to target Src pathways in premalignant breast lesions to limit the development of breast cancers. Cancer Res; 75(22); 4863–75. ©2015 AACR.

RPPA data showing fold changes in transcriptional regulator expressions in saracatinib treated 10A.B2 cells over vehicle control.

Description of additional methods and procedures used in the study. Also includes Supplementary References.

Figure S4. Primary tumor incidence, growth, survival and metastasis in Ptenf/f vs. Arf6Q67L mice. (a) Increased tumor incidence (% = # of mice that developed a tumor / # TVA positive, injected mice) in Ptenf/f vs. Arf6Q67L cohorts. Fisher's exact test, two-tailed, α

Melanoma has an unusual capacity to spread in early-stage disease, prompting aggressive clinical intervention in very thin primary tumors. Despite these proactive efforts, patients with low-risk, low-stage disease can still develop metastasis, indicating the presence of permissive cues for distant spread. Here, we show that constitutive activation of the small GTPase ARF6 (ARF6Q67L) is sufficient to accelerate metastasis in mice with BRAFV600E/Cdkn2aNULL melanoma at a similar incidence and severity to Pten loss, a major driver of PI3K activation and melanoma metastasis. ARF6Q67L promoted spontaneous metastasis from significantly smaller primary tumors than PTENNULL, implying an enhanced ability of ARF6-GTP to drive distant spread. ARF6 activation increased lung colonization from circulating melanoma cells, suggesting that the prometastatic function of ARF6 extends to late steps in metastasis. Unexpectedly, ARF6Q67L tumors showed upregulation of Pik3r1 expression, which encodes the p85 regulatory subunit of PI3K. Tumor cells expressing ARF6Q67L displayed increased PI3K protein levels and activity, enhanced PI3K distribution to cellular protrusions, and increased AKT activation in invadopodia. ARF6 is necessary and sufficient for activation of both PI3K and AKT, and PI3K and AKT are necessary for ARF6-mediated invasion. We provide evidence for aberrant ARF6 activation in human melanoma samples, which is associated with reduced survival. Our work reveals a previously unknown ARF6-PI3K-AKT proinvasive pathway, it demonstrates a critical role for ARF6 in multiple steps of the metastatic cascade, and it illuminates how melanoma cells can acquire an early metastatic phenotype in patients.Significance:These findings reveal a prometastatic role for ARF6 independent of tumor growth, which may help explain how melanoma spreads distantly from thin, early-stage primary tumors.

Table S1: ARF6 pathways and related genes.

Legend for Supplementary Table S1 and Supplementary Figures S1-S5.

PDF file - 255K, The observation of more lymphocytes/leukocytes infiltration after HER2/Neu antibody and Akt inhibitor TCN combination treatment

PDF file - 206K, No significant changes in the number of tumor infiltrating T cells (including CD8+ and CD4+ ) upon the addition of anti-CTLA-4 mAb to HER2/Neu antibody and triciribine combination treatment

PDF file - 25K, HER2/Neu antibody 7.16.4 and Akt inhibitor TCN combination treatment reduced tumor multiplicity and lung metastasis in PTEN-/-/NIC mice

PDF file - 410K, Histological and immunohistochemical analysis of tumors derived from PTEN-/-/ErbB2KI mice receiving single or combination treatment

PDF file - 137K, Depletion of CD8+ cells, CD4+ cells or both during HER2/Neu antibody and triciribine combination treatment

Trastuzumab is an iconic rationally designed targeted therapy for HER2-positive breast cancers. However, the low response rate and development of resistance call for novel approaches for the treatment of patients. Here, we report that concurrent targeting of tumor cells and activation of T cells in the tumor microenvironment results in a synergistic inhibitory effect on tumor growth and overcomes resistance in two distinct PTEN loss–mediated trastuzumab-resistant mammary tumor mouse models. In vivo combination treatment with HER2/Neu antibody and Akt inhibitor triciribine effectively inhibited tumor growth in both models via inhibiting PI3K/AKT and mitogen-activated protein kinase signaling accompanied by increased T-cell infiltration in the tumor microenvironment. We showed that both CD8+ and CD4+ T cells were essential to the optimal antitumor effect of this combination treatment in an IFN-γ–dependent manner. Importantly, the antitumor activities of HER2/Neu antibody and triciribine combination treatment were further improved when coinhibitory receptor cytotoxic T-lymphocyte–associated antigen 4 was blocked to enhance the T-cell response. Our data indicate that multitargeted combinatorial therapies targeting tumor cells and concomitantly enhancing T-cell response in the tumor microenvironment could cooperate to exert maximal therapeutic activity, suggesting a promising clinical strategy for treating trastuzumab-resistant breast cancers and other advanced malignancies. Cancer Res; 72(17); 4417–28. ©2012 AACR.

PDF file - 24K, HER2/Neu antibody and TCN plus anti-CTLA-4 mAb triple combination treatment increased serum IFN-gamma level

PDF file - 37K, Anti-CTLA-4 mAb alone, anti-CTLA-4 mAb combined with HER2/Neu antibody only, or with TCN only showed no significant enhancement of antitumor effect

PDF file - 82K, Primer sequences for quantitative real-time reverse transcription-PCR

PDF file - 85K, Characterization of PTEN-/-/NIC mouse model

PDF file - 126K

PDF file - 36K, Evaluation of markers of other tumor-infiltrating immune cells

Glucocorticoids (GCs) are a cornerstone of acute lymphoblastic leukemia (ALL) therapy. While mutations in NR3C1, which encodes the GC receptor (GR), and other genes involved in GC signaling occur at relapse, additional mechanisms of adaptive GC resistance are uncertain. We transplanted and treated ten primary mouse T-lineage acute lymphoblastic leukemias (T-ALLs) initiated by retroviral insertional mutagenesis with the GC dexamethasone (DEX). Multiple, distinct relapsed clones from one such leukemia (T-ALL 8633) exhibited discrete retroviral integrations that up-regulated Jdp2 expression. This leukemia harbored a Kdm6a mutation. In the human T-ALL cell line CCRF-CEM, enforced JDP2 over-expression conferred GC resistance, while KDM6A inactivation unexpectedly enhanced GC sensitivity. In the context of KDM6A knock-out, JDP2 over-expression induced profound GC resistance, counteracting the sensitization conferred by KDM6A loss. These resistant "double mutant" cells with combined KDM6A loss and JDP2 over-expression exhibited decreased NR3C1 mRNA and GR protein up-regulation upon DEX exposure. Analysis of paired samples from two KDM6A-mutant T-ALL patients in a relapsed pediatric ALL cohort revealed a somatic NR3C1 mutation at relapse in one and markedly elevated JDP2 expression in another. Together, these data implicate JDP2 over-expression as a mechanism of adaptive GC resistance in T-ALL that functionally interacts with KDM6A inactivation.

Mutational signature analysis is commonly performed in cancer genomic studies. Here, we present SigProfilerExtractor, an automated tool for de novo extraction of mutational signatures, and benchmark it against another 13 bioinformatics tools by using 34 scenarios encompassing 2,500 simulated signatures found in 60,000 synthetic genomes and 20,000 synthetic exomes. For simulations with 5% noise, reflecting high-quality datasets, SigProfilerExtractor outperforms other approaches by elucidating between 20% and 50% more true-positive signatures while yielding 5-fold less false-positive signatures. Applying SigProfilerExtractor to 4,643 whole-genome- and 19,184 whole-exome-sequenced cancers reveals four novel signatures. Two of the signatures are confirmed in independent cohorts, and one of these signatures is associated with tobacco smoking. In summary, this report provides a reference tool for analysis of mutational signatures, a comprehensive benchmarking of bioinformatics tools for extracting signatures, and several novel mutational signatures, including one putatively attributed to direct tobacco smoking mutagenesis in bladder tissues. published

Chemotherapy is a standard treatment for pediatric acute lymphoblastic leukemia (ALL), which sometimes relapses with chemoresistant features. However, whether acquired drug-resistance mutations in relapsed ALL pre-exist or are induced by treatment remains unknown. Here we provide direct evidence of a specific mechanism by which chemotherapy induces drug-resistance-associated mutations leading to relapse. Using genomic and functional analysis of relapsed ALL we show that thiopurine treatment in mismatch repair (MMR)-deficient leukemias induces hotspot TP53 R248Q mutations through a specific mutational signature (thio-dMMR). Clonal evolution analysis reveals sequential MMR inactivation followed by TP53 mutation in some patients with ALL. Acquired TP53 R248Q mutations are associated with on-treatment relapse, poor treatment response and resistance to multiple chemotherapeutic agents, which could be reversed by pharmacological p53 reactivation. Our findings indicate that TP53 R248Q in relapsed ALL originates through synergistic mutagenesis from thiopurine treatment and MMR deficiency and suggest strategies to prevent or treat TP53-mutant relapse. Zhou and colleagues demonstrate that thiopurine chemotherapy in mismatch repair-deficient ALL cells leads to R248Q TP53 mutations and clonal selection that favors on-treatment ALL relapse and chemoresistance.

Pediatric therapy-related myeloid neoplasms (tMN) occur in children after exposure to cytotoxic therapy and have a dismal prognosis. The somatic and germline genomic alterations that drive these myeloid neoplasms in children and how they arise have yet to be comprehensively described. We use whole exome, whole genome, and/or RNA sequencing to characterize the genomic profile of 84 pediatric tMN cases (tMDS: n = 28, tAML: n = 56). Our data show that Ras/MAPK pathway mutations, alterations in RUNX1 or TP53, and KMT2A rearrangements are frequent somatic drivers, and we identify cases with aberrant MECOM expression secondary to enhancer hijacking. Unlike adults with tMN, we find no evidence of pre-existing minor tMN clones (including those with TP53 mutations), but rather the majority of cases are unrelated clones arising as a consequence of cytotoxic therapy. These studies also uncover rare cases of lineage switch disease rather than true secondary neoplasms., Paediatric therapy-related myeloid neoplasms (tMN) have a dismal prognosis and have not been comprehensively profiled. Here the authors characterise the molecular landscape of 84 paediatric tMN patients, and find that, unlike adult tMNs, these do not emerge from pre-existing clones and that MECOM dysregulation is frequent.

Effective data sharing is key to accelerating research to improve diagnostic precision, treatment efficacy, and long-term survival in pediatric cancer and other childhood catastrophic diseases. We present St. Jude Cloud (https://www.stjude.cloud), a cloud-based data-sharing ecosystem for accessing, analyzing, and visualizing genomic data from >10,000 pediatric patients with cancer and long-term survivors, and >800 pediatric sickle cell patients. Harmonized genomic data totaling 1.25 petabytes are freely available, including 12,104 whole genomes, 7,697 whole exomes, and 2,202 transcriptomes. The resource is expanding rapidly, with regular data uploads from St. Jude's prospective clinical genomics programs. Three interconnected apps within the ecosystem—Genomics Platform, Pediatric Cancer Knowledgebase, and Visualization Community—enable simultaneously performing advanced data analysis in the cloud and enhancing the Pediatric Cancer knowledgebase. We demonstrate the value of the ecosystem through use cases that classify 135 pediatric cancer subtypes by gene expression profiling and map mutational signatures across 35 pediatric cancer subtypes. Significance: To advance research and treatment of pediatric cancer, we developed St. Jude Cloud, a data-sharing ecosystem for accessing >1.2 petabytes of raw genomic data from >10,000 pediatric patients and survivors, innovative analysis workflows, integrative multiomics visualizations, and a knowledgebase of published data contributed by the global pediatric cancer community. This article is highlighted in the In This Issue feature, p. 995

The somatic mutations in each cancer genome are caused by multiple mutational processes, each of which leaves a characteristic imprint (or 'signature'), potentially caused by specific etiologies or exposures. Deconvolution of these signatures offers a glimpse into the evolutionary history of individual tumors. Recent work has shown that mutational signatures may also yield therapeutic and prognostic insights, including the identification of cell-intrinsic signatures as biomarkers of drug response and prognosis. For example, mutational signatures indicating homologous recombination deficiency are associated with poly(ADP)-ribose polymerase (PARP) inhibitor sensitivity, whereas APOBEC-associated signatures are associated with ataxia telangiectasia and Rad3-related kinase (ATR) inhibitor sensitivity. Furthermore, therapy-induced mutational signatures implicated in cancer progression have also been uncovered, including the identification of thiopurine-induced TP53 mutations in leukemia. In this review, we explore the various ways mutational signatures can reveal new therapeutic and prognostic insights, thus extending their traditional role in identifying disease etiology.

To study the mechanisms of relapse in acute lymphoblastic leukemia (ALL), we performed whole-genome sequencing of 103 diagnosis-relapse-germline trios and ultra-deep sequencing of 208 serial samples in 16 patients. Relapse-specific somatic alterations were enriched in 12 genes (NR3C1, NR3C2, TP53, NT5C2, FPGS, CREBBP, MSH2, MSH6, PMS2, WHSC1, PRPS1, and PRPS2) involved in drug response. Their prevalence was 17% in very early relapse (36 months) groups. Convergent evolution, in which multiple subclones harbor mutations in the same drug resistance gene, was observed in 6 relapses and confirmed by single-cell sequencing in 1 case. Mathematical modeling and mutational signature analysis indicated that early relapse resistance acquisition was frequently a 2-step process in which a persistent clone survived initial therapy and later acquired bona fide resistance mutations during therapy. In contrast, very early relapses arose from preexisting resistant clone(s). Two novel relapse-specific mutational signatures, one of which was caused by thiopurine treatment based on in vitro drug exposure experiments, were identified in early and late relapses but were absent from 2540 pan-cancer diagnosis samples and 129 non-ALL relapses. The novel signatures were detected in 27% of relapsed ALLs and were responsible for 46% of acquired resistance mutations in NT5C2, PRPS1, NR3C1, and TP53. These results suggest that chemotherapy-induced drug resistance mutations facilitate a subset of pediatric ALL relapses.