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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

Supplementary Figure S2

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

Supplementary Figure S6

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.

Supplementary Figure S5

Supplementary Figure S1

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.

Supplementary Figure S7

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.

Supplementary Figure S3

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

Effect of Src on Myc transcription, protein stability and synthesis.

Saracatinib increases tumor free and overall survival in ER- mammary mouse models.

Figure S7. Low expression of ARF GAP genes correlates with reduced overall survival. (a) Individual gene plots of four ARF6 GAPs (ACAP1, ADAP1, ARAP2, SMAP2), an ARF1/ARF5 GAP (AGAP2) and one predicted ARF GAP (AGFG2). n = 439 patients. (b) Individual gene plots in Stage III specific cohort, n=142 patients. Log-rank (Mantel-Cox) test.

Figure S6. ARF6-GDP inhibits PI3K-AKT activation and is necessary for AKT activation. (a-b) Quantification of western blots shown in Figure 5c-d. Reproducible reduction in pAKT by Myc-ARF6T27N over expression in A375 cells (n=4), A2058 cells (n=4), and HEY-T30 cells (n=5). Reduced pAKT with siRNA knockdown of ARF6 in SK-MEL-147 (n=5), A2058 (n=4), and CACL (n=4) and HEY-T30 (n=6). Graphs show individual data points normalized to control along with geometric means, 95% CI, ratio paired t test. (c) Phosphokinase array images (left) and quantification of signal by densitometry (right, histogram), n=1. Expression of ARF6T27N in A2058 cells preferentially reduced pAKT and the AKT substrate PRAS40 over most other proteins in the array.

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.

Figure S1. Detection of HA-tagged ARF6Q67L in BrafCA;Cdkn2af/f melanoma. a) Anti-HA immunostain of FFPE sections of primary tumors with variable levels of detection of ARF6Q67L -HA. The strongest staining was observed in tumors with the highest level of mRNA detected by qRT-PCR (b). Absent staining of FFPE tumors was observed in tumors with low (6456) and undetectable (6457) levels of mRNA by qRT-PCR of frozen tumors. Scale bars = 20mm, 400x magnification. Control = BrafCA;Cdkn2af/f (Cre-only injection). b) qRT-PCR of Arf6 Q67L-HA from fresh, frozen primary tumor fragments. Bars represent the absolute difference in crossing threshold (Ct) between Arf6Q67L-HA and Gapdh. The Ct threshold that defines detection was established above the background signal for controls in both the BrafCA;Cdkn2af/f and the BrafCA;Cdkn2af/f ;Ptenf/f cohorts (see Figures 1g and S3). With rare exception, positive tumors demonstrated Arf6 Q67L-HA expression levels lower than Gapdh.

Table S1: ARF6 pathways and related genes.

Figure S5: ARF6-GTP induces morphologic changes in mouse melanoma cells. Bright field images (100x magnification) of primary mouse melanoma cell lines derived from control BrafCA;Cdkn2af/f (5588) or BrafCA;Cdkn2af/f + Arf6Q67L (6431, 6455) mice. ARF6-GTP induces elongation/spindling of tumor cells relative to the more polygonal-shaped controls. Scale bars = 400um.

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

Targeting Src prevents the development of disorganized acinar growth.

Src regulates GLUT1 expression.

Figure S3. Arf6Q67L-HA expression in tumors from BrafCA;Cdkn2af/f;Ptenf/f mice. qRT-PCR detection of Arf6 Q67L-HA from fresh, frozen primary tumor fragments. Bars represent the absolute difference in crossing threshold (Ct) between Arf6 Q67L-HA and Gapdh. The Ct threshold that defines detection was established above the background signal for controls in both the BrafCA;Cdkn2af/f and the BrafCA;Cdkn2af/f ;Ptenf/f cohorts (see also Figure S1). All mouse tumor negative by qRT-PCR showed detectable HA-tagged ARF6Q67L by immunohistochemistry (not shown, see Figure S1 for example).

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

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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.

Background Metastatic breast cancer is a deadly disease with a low 5-year survival rate. Tracking metastatic spread in living patients is difficult and thus poorly understood. Methods Via rapid autopsy, we have collected 30 tumor samples over 3 timepoints and across 8 organs from a triple-negative metastatic breast cancer patient. The large number of sites sampled, together with deep whole-genome sequencing and advanced computational analysis, allowed us to comprehensively reconstruct the tumor’s evolution at subclonal resolution. Results The most unique, previously unreported aspect of the tumor’s evolution that we observed in this patient was the presence of “subclone incubators,” defined as metastatic sites where substantial tumor evolution occurs before colonization of additional sites and organs by subclones that initially evolved at the incubator site. Overall, we identified four discrete waves of metastatic expansions, each of which resulted in a number of new, genetically similar metastasis sites that also enriched for particular organs (e.g., abdominal vs bone and brain). The lung played a critical role in facilitating metastatic spread in this patient: the lung was the first site of metastatic escape from the primary breast lesion, subclones at this site were likely the source of all four subsequent metastatic waves, and multiple sites in the lung acted as subclone incubators. Finally, functional annotation revealed that many known drivers or metastasis-promoting tumor mutations in this patient were shared by some, but not all metastatic sites, highlighting the need for more comprehensive surveys of a patient’s metastases for effective clinical intervention. Conclusions Our analysis revealed the presence of substantial tumor evolution at metastatic incubator sites in a patient, with potentially important clinical implications. Our study demonstrated that sampling of a large number of metastatic sites affords unprecedented detail for studying metastatic evolution.