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A quantum computer attains computational advantage when outperforming the best classical computers running the best-known algorithms on well-defined tasks. No photonic machine offering programmability over all its quantum gates has demonstrated quantum computational advantage: previous machines1,2 were largely restricted to static gate sequences. Earlier photonic demonstrations were also vulnerable to spoofing3, in which classical heuristics produce samples, without direct simulation, lying closer to the ideal distribution than do samples from the quantum hardware. Here we report quantum computational advantage using Borealis, a photonic processor offering dynamic programmability on all gates implemented. We carry out Gaussian boson sampling4 (GBS) on 216 squeezed modes entangled with three-dimensional connectivity5, using a time-multiplexed and photon-number-resolving architecture. On average, it would take more than 9,000 years for the best available algorithms and supercomputers to produce, using exact methods, a single sample from the programmed distribution, whereas Borealis requires only 36 μs. This runtime advantage is over 50 million times as extreme as that reported from earlier photonic machines. Ours constitutes a very large GBS experiment, registering events with up to 219 photons and a mean photon number of 125. This work is a critical milestone on the path to a practical quantum computer, validating key technological features of photonics as a platform for this goal.

Supplementary Figure S1. ALK Tg mice develop lung multifocal adenocarcinomas similar to human ALK-rearranged NSCLC. Supplementary Figure S2. Therapeutic ALK vaccine impairs tumor growth of ALK-rearranged lung tumors. Supplementary Figure S3. Immunophenotype of EML4-ALK transgenic mice Supplementary Figure S4. Human ALK-rearranged NSCLC show a pattern of low expression of T cell markers. Supplementary Figure S5. PD-L1 expression is dependent on ALK kinase activity in ALK-rearranged NSCLC cells. Supplementary Figure S6. Schematic protocols of the in vivo treatment with anti-PD-1 blocking antibody. Supplementary Figure S7. ALK vaccine can be combined with ALK inhibitor TAE684. Supplementary Figure S8. Immunophenotype of intratumoral T cells in ALK vaccinated mice.

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Supplementary Materials and Methods

Non–small cell lung cancer (NSCLC) harboring chromosomal rearrangements of the anaplastic lymphoma kinase (ALK) gene is treated with ALK tyrosine kinase inhibitors (TKI), but the treatment is successful for only a limited amount of time; most patients experience a relapse due to the development of drug resistance. Here, we show that a vaccine against ALK induced a strong and specific immune response that both prophylactically and therapeutically impaired the growth of ALK-positive lung tumors in mouse models. The ALK vaccine was efficacious also in combination with ALK TKI treatment and significantly delayed tumor relapses after TKI suspension. We found that lung tumors containing ALK rearrangements induced an immunosuppressive microenvironment, regulating the expression of PD-L1 on the surface of lung tumor cells. High PD-L1 expression reduced ALK vaccine efficacy, which could be restored by administration of anti–PD-1 immunotherapy. Thus, combinations of ALK vaccine with TKIs and immune checkpoint blockade therapies might represent a powerful strategy for the treatment of ALK-driven NSCLC. Cancer Immunol Res; 3(12); 1333–43. ©2015 AACR.

Supplementary Figures 1-19. Supplementary Figure 1. Hypoxia pathways are enriched in ALCL. Supplementary Figure 2. Gene expression data on ALCL. Supplementary Figure 3. EML4-ALK NSCLC show enrichment in hypoxia pathway associated genes. Supplementary Figure 4. ALK regulates HIF-1α and HIF-2α expression in ALK-rearranged ALCL cell lines. Supplementary Figure 5. Oncogenic ALK regulates HIF-1α and HIF-2α expression levels in ALK-rearranged ALCL cell lines. Supplementary Figure 6. Oncogenic ALK-mediated control of HIF-1α and HIF-2α expression is not dependent on proteasome-dependent degradation. Supplementary Figure 7. ALK regulates HIF-1α and HIF-2α through STAT3 and C/EBPβ. Supplementary Figure 8. STAT3 and C/EBPβ mediated regulation of HIFs in ALCL. Supplementary Figure 9. Ectopic expression of NPM-ALK in ALK negative ALCL. Supplementary Figure 10. Validation of shRNA against HIF-1α and HIF-2α. Supplementary Figure 11. HIF-2α is essential for the growth of ALK-rearranged ALCL in vivo. Supplementary Figure 12. HIF-1α and HIF-2α are not required for the growth of ALK-negative T cell lymphoma in vivo. Supplementary Figure 13. ALK regulates VEGFA mRNA expression and production in vitro. Supplementary Figure 14. Anti-angiogenic treatment in ALK negative ALCL. Supplementary Figure 15. Oncogenic ALK regulates HIF-1α and HIF-2α expression levels in EML4-ALK NSCLC. Supplementary Figure 16. HIF-α regulation in NSCLC is specifically dependent on ALK activity. Supplementary Figure 17. Oncogenic ALK activity up-regulates VEGF expression in NSCLC. Supplementary Figure 18. HIF-α knock-down does not affect the growth of K-RAS mutated NSCLC. Supplementary Figure 19. HIF-1α and HIF-2α are essential for metastasis formation in EML4-ALK NSCLC.

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Fibroblasts display extensive transcriptional heterogeneity, yet functional annotation and characterization of their heterocellular relationships remains incomplete. Using mass cytometry, we chart the stromal composition of 18 murine tissues and 5 spontaneous tumor models, with an emphasis on mesenchymal phenotypes. This analysis reveals extensive stromal heterogeneity across tissues and tumors, and identifies novel, coordinated, relationships between mesenchymal and immune cell subsets. Expression of CD105 demarks two stable and functionally distinct fibroblast lineages, that are ubiquitous in murine and human healthy tissues and tumors. Whereas CD105 positive fibroblasts are permissive for tumor growth in vivo, CD105 negative fibroblasts are highly tumor suppressive. This restrictive effect is entirely dependent on functional adaptive immunity, with a major contribution from type 1 conventional dendritic cells. Collectively, these results reveal two distinct fibroblast lineages conserved across tissues and species and highlight the importance of mesenchymal and immune cell interactions in restricting tumor growth., This upload consists of raw data as analysed in Hutton et al.

Summary Fibroblasts display extensive transcriptional heterogeneity, yet functional annotation and characterization of their heterocellular relationships remains incomplete. Using mass cytometry, we chart the stromal composition of 18 murine tissues and 5 spontaneous tumor models, with an emphasis on mesenchymal phenotypes. This analysis reveals extensive stromal heterogeneity across tissues and tumors, and identifies coordinated relationships between mesenchymal and immune cell subsets in pancreatic ductal adenocarcinoma. Expression of CD105 demarks two stable and functionally distinct pancreatic fibroblast lineages, which are also identified in murine and human healthy tissues and tumors. Whereas CD105-positive pancreatic fibroblasts are permissive for tumor growth in vivo, CD105-negative fibroblasts are highly tumor suppressive. This restrictive effect is entirely dependent on functional adaptive immunity. Collectively, these results reveal two functionally distinct pancreatic fibroblast lineages and highlight the importance of mesenchymal and immune cell interactions in restricting tumor growth., Graphical abstract, Highlights • Mass cytometry analysis of mesenchymal stroma in murine normal and tumor tissue • Mesenchymal heterogeneity is a feature of human and murine tissues and tumors • CD105 expression distinguishes two pancreatic fibroblast lineages • CD105neg pancreatic fibroblasts support anti-tumor immunity to control tumor growth, Hutton et al. use mass cytometry to chart stromal cells and describe mesenchymal states and lineages in pancreatic ductal adenocarcinoma. CD105 (Eng) expression distinguishes two pancreatic fibroblast lineages with distinct functions. CD105pos fibroblasts are tumor permissive, whereas CD105neg fibroblasts suppress tumor growth in a manner dependent on adaptive immunity.

In T lymphocytes, the Wiskott–Aldrich Syndrome protein (WASP) and WASP-interacting-protein (WIP) regulate T cell antigen receptor (TCR) signaling, but their role in lymphoma is largely unknown. Here we show that the expression of WASP and WIP is frequently low or absent in anaplastic large cell lymphoma (ALCL) compared to other T cell lymphomas. In anaplastic lymphoma kinase–positive (ALK+) ALCL, WASP and WIP expression is regulated by ALK oncogenic activity via its downstream mediators STAT3 and C/EBP-β. ALK+ lymphomas were accelerated in WASP- and WIP-deficient mice. In the absence of WASP, active GTP-bound CDC42 was increased and the genetic deletion of one CDC42 allele was sufficient to impair lymphoma growth. WASP-deficient lymphoma showed increased mitogen-activated protein kinase (MAPK) pathway activation that could be exploited as a therapeutic vulnerability. Our findings demonstrate that WASP and WIP are tumor suppressors in T cell lymphoma and suggest that MAP-kinase kinase (MEK) inhibitors combined with ALK inhibitors could achieve a more potent therapeutic effect in ALK+ ALCL., The work has been supported by grant no. FP7 ERC-2009-StG (Proposal No. 242965—‘Lunely’) (R.C.) grant no. R01 CA196703-01 (R.C.); AIRC grant no. MFAG (C.A. and M.C.); National Research Foundation of Korea (NRF) fellowship 2016R1A6A3A03006840 (T-C.C.); Bando Giovani Ricercatori grant no. 2009-GR 1603126 (M.C.); MINECO/FEDER grant no. SAF2015–70368-R and Fundación Ramón Areces (I.M.A.); the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health (L.D.N.); and award no. T32GM007753 from the National Institute of General Medical Sciences (S.H.C.) (the content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health); and in part by awards from the National Institutes of Health DP2 New Innovator award no. 1DP2CA195762-01 (C.K.); the American Cancer Society Research Scholar award no. RSG-14-051-01-DMC and the Pew-Stewart Scholars in Cancer Research Grant (C.K.); and the European Union Horizon 2020 Marie Sklodowska-Curie Innovative Training Network Grant award no. 675712 for the European Research Initiative for ALK-Related Malignancies (G.G.S., I.M., C.GP. and R.C.).

Rearrangements involving the anaplastic lymphoma kinase (ALK) gene are defining events in several tumors, including anaplastic large-cell lymphoma (ALCL) and non–small cell lung carcinoma (NSCLC). In such cancers, the oncogenic activity of ALK stimulates signaling pathways that induce cell transformation and promote tumor growth. In search for common pathways activated by oncogenic ALK across different tumors types, we found that hypoxia pathways were significantly enriched in ALK-rearranged ALCL and NSCLC, as compared with other types of T-cell lymphoma or EGFR- and K-RAS–mutated NSCLC, respectively. Consistently, in both ALCL and NSCLC, we found that under hypoxic conditions, ALK directly regulated the abundance of hypoxia-inducible factors (HIF), which are key players of the hypoxia response in normal tissues and cancers. In ALCL, the upregulation of HIF1α and HIF2α in hypoxic conditions required ALK activity and its downstream signaling proteins STAT3 and C/EBPβ. In vivo, ALK regulated VEGFA production and tumor angiogenesis in ALCL and NSCLC, and the treatment with the anti-VEGFA antibody bevacizumab strongly impaired ALCL growth in mouse xenografts. Finally, HIF2α, but not HIF1α, was required for ALCL growth in vivo whereas the growth and metastasis potential of ALK-rearranged NSCLC required both HIF1α and HIF2α. In conclusion, we uncovered an ALK-specific regulation of the hypoxia response across different ALK+ tumor types and propose HIFs as a powerful specific therapeutic target in ALK-rearranged ALCL and NSCLC. Cancer Res; 74(21); 6094–106. ©2014 AACR.

Most of the anaplastic large-cell lymphoma (ALCL) cases carry the t(2;5; p23;q35) that produces the fusion protein NPM-ALK (nucleophosmin-anaplastic lymphoma kinase). NPM-ALK-deregulated kinase activity drives several pathways that support malignant transformation of lymphoma cells. We found that in ALK-rearranged ALCL cell lines, NPM-ALK was distributed in equal amounts between the cytoplasm and the nucleus. Only the cytoplasmic portion was catalytically active in both cell lines and primary ALCL, whereas the nuclear portion was inactive because of heterodimerization with NPM1. Thus, about 50% of the NPM-ALK is not active and sequestered as NPM-ALK/NPM1 heterodimers in the nucleus. Overexpression or relocalization of NPM-ALK to the cytoplasm by NPM genetic knockout or knockdown caused ERK1/2 (extracellular signal-regulated protein kinases 1 and 2) increased phosphorylation and cell death through the engagement of an ATM/Chk2- and γH2AX (phosphorylated H2A histone family member X)-mediated DNA-damage response. Remarkably, human NPM-ALK-amplified cell lines resistant to ALK tyrosine kinase inhibitors (TKIs) underwent apoptosis upon drug withdrawal as a consequence of ERK1/2 hyperactivation. Altogether, these findings indicate that an excess of NPM-ALK activation and signaling induces apoptosis via oncogenic stress responses. A 'drug holiday' where the ALK TKI treatment is suspended could represent a therapeutic option in cells that become resistant by NPM-ALK amplification.

Increasing evidence suggests that Rho family GTPases could have a critical role in the biology of T-cell lymphoma. In ALK-rearranged anaplastic large cell lymphoma (ALCL), a specific subtype of T-cell lymphoma, the Rho family GTPases Cdc42 and Rac1 are activated by the ALK oncogenic activity. In vitro studies have shown that Cdc42 and Rac1 control rather similar phenotypes of ALCL biology such as the proliferation, survival, and migration of lymphoma cells. However, their role and possible redundancy in ALK-driven lymphoma development in vivo are still undetermined. We genetically deleted Cdc42 or Rac1 in a mouse model of ALK-rearranged ALCL to show that either Cdc42 or Rac1 deletion impaired lymphoma development, modified lymphoma morphology, actin filament distribution, and migration properties of lymphoma cells. Cdc42 or Rac1 deletion primarily affected survival rather than proliferation of lymphoma cells. Apoptosis of lymphoma cells was equally induced following Cdc42 or Rac1 deletion, was associated with upregulation of the proapoptotic molecule Bid, and was blocked by Bcl2 overexpression. Remarkably, Cdc42/Rac1 double deletion, but not Cdc42 or Rac1 single deletions, completely prevented NPM-ALK lymphoma dissemination in vivo. Thus, Cdc42 and Rac1 have nonredundant roles in controlling ALK-rearranged lymphoma survival and morphology but are redundant for lymphoma dissemination, suggesting that targeting both GTPases could represent a preferable therapeutic option for ALCL treatment.

A subset of Non-Small Cell Lung Carcinoma (NSCLC) carries chromosomal rearrangements involving the Anaplastic Lymphoma Kinase (ALK) gene. ALK-rearranged NSCLC are typically adenocarcinoma characterized by a solid signet-ring cell pattern that is frequently associated with a metastatic phenotype. Recent reports linked the presence of ALK rearrangement to an epithelial-mesenchymal transition (EMT) phenotype in NSCLC, but the extent and the mechanisms of an ALK-mediated EMT in ALK-rearranged NSCLC are largely unknown. We found that the ALK-rearranged H2228 and DFCI032, but not the H3122, cell lines displayed a mesenchymal phenotype. In these cell lines, oncogenic ALK activity dictated an EMT phenotype by directly suppressing E-cadherin and up-regulating vimentin expression, as well as expression of other genes involved in EMT. We found that the epithelial splicing regulatory protein 1 (ESRP1), a key regulator of the splicing switch during EMT, was repressed by EML4-ALK activity. The treatment of NSCLC cells with ALK tyrosine kinase inhibitors (TKIs) led to up-regulation of ESRP1 and E-cadherin, thus reverting the phenotype from mesenchymal to epithelial (MET). Consistently, ESRP1 knock-down impaired E-cadherin up-regulation upon ALK inhibition, whereas enforced expression of ESRP1 was sufficient to increase E-cadherin expression. These findings demonstrate an ALK oncogenic activity in the regulation of an EMT phenotype in a subset of NSCLC with potential implications for the biology of ALK-rearranged NSCLC in terms of metastatic propensity and resistance to therapy.

Non–small cell lung cancer (NSCLC) harboring chromosomal rearrangements of the anaplastic lymphoma kinase (ALK) gene is treated with ALK tyrosine kinase inhibitors (TKI), but the treatment is successful for only a limited amount of time; most patients experience a relapse due to the development of drug resistance. Here, we show that a vaccine against ALK induced a strong and specific immune response that both prophylactically and therapeutically impaired the growth of ALK-positive lung tumors in mouse models. The ALK vaccine was efficacious also in combination with ALK TKI treatment and significantly delayed tumor relapses after TKI suspension. We found that lung tumors containing ALK rearrangements induced an immunosuppressive microenvironment, regulating the expression of PD-L1 on the surface of lung tumor cells. High PD-L1 expression reduced ALK vaccine efficacy, which could be restored by administration of anti–PD-1 immunotherapy. Thus, combinations of ALK vaccine with TKIs and immune checkpoint blockade therapies might represent a powerful strategy for the treatment of ALK-driven NSCLC. Cancer Immunol Res; 3(12); 1333–43. ©2015 AACR.

Most of Anaplastic Large Cell Lymphoma (ALCL) cases carry the t(2;5; p23;q35) that produces the fusion protein nucleophosmin (NPM)-ALK. NPM provides an oligomerization domain that causes the spontaneous dimerization and ligand-independent activation of the ALK. NPM-ALK deregulated kinase activity drives several pathways involved in cellular proliferation and survival and sustains the process of malignant transformation. NPM-ALK is localized both in the cytoplasm and the nucleus, but the role of NPM-ALK cytoplasmic or nuclear fractions on ALCL phenotype maintenance is undetermined. We found that in all ALK+ ALCL cell lines and in primary ALCL NPM-ALK was equally distributed between cytoplasm and nucleus, but only the cytoplasmic portion, formed by NPM-ALK homodimers, was kinetically active and had transforming properties. The nuclear portion, formed by NPM/NPM-ALK heterodimers, was inactive. Thus, about 50% of the NPM-ALK fusion is sequestered and inactivated in the nucleus of ALCL cells by WT NPM1. Indeed, relocalization of NPM-ALK entirely to the cytoplasm in NPM-/- cells or its overexpression in several ALK+ ALCL cell lines resulted in cell death through activation of the ERK1/2 pathway and of γ-H2AX, a key player of the DNA damage response pathway. Thus, excessive oncogenic ALK signaling induces a cell stress leading to apoptosis and a balanced amount of ALK activation is necessary for optimal ALCL growth. Interestingly, we selected three human NPM-ALK positive cell lines resistant to the dual ALK/EGFR inhibitor AP26113. Resistance was due to genomic NPM-ALK amplification that caused overexpression of NPM-ALK and consequently hyperactivation of downstream ALK-dependent pathways. These cell lines were not only resistant, but also drug-dependent, meaning that addition of a low amount of AP26113 was required for cellular growth and to keep “normal” NPM-ALK signalling levels. Indeed, upon drug withdrawal, NPM-ALK and its downstream pathways were hyperactivated. The viability of all three drug-addicted cell lines dramatically dropped, confirming that an excess of NPM-ALK signaling was detrimental for cell growth. In two resistant cell lines out of three a new population recovered in the absence of the drug and was re-sensitized to AP26113. Notably, in these new drug-sensitive populations NPM-ALK overexpression was abrogated. All together, these findings support the idea that deregulation of NPM-ALK localization might be an attractive therapeutic option. Moreover, for a subset of patients that may develop drug dependency as well as drug resistance as a consequence of NPM-ALK overexpression, a discontinuous tyrosine kinase inhibitor therapeutic schedule may be more effective than a continuous one, as it is usually proposed. Citation Format: Monica Ceccon, Maria Elena Boggio Merlo, Luca Mologni, Lydia Varesio, Teresa Poggio, Matteo Menotti, Silvia Bombelli, Roberta Rigolio, Andrea Manazza, Chiara Ambrogio, Giovanni Giudici, Cesare Casati, Mara Compagno, Suzanne Turner, Carlo Gambacorti Passerini, Roberto Chiarle, Claudia Voena. Excess of cytoplasmic NPM-ALK driven oncogenic signaling is toxic and promotes cellular apoptosis and drug dependency. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2920. doi:10.1158/1538-7445.AM2015-2920

A subset of Non-Small Cell Lung Carcinoma (NSCLC) carries chromosomal rearrangements involving the Anaplastic Lymphoma Kinase (ALK) gene. More frequently ALK is juxtaposed to the echinoderm microtubule-associated protein-like 4 (EML4) gene on chromosome 2 and generates a constitutively active EML4-ALK fusion protein that triggers downstream oncogenic signals leading to increased cell proliferation and survival. The majority of ALK-rearranged NSCLC presents a peculiar histology characterized by a solid signet-ring cell and a mucinous cribriform pattern that is frequently associated with a metastatic phenotype. As the signet ring phenotype and metastasis are associated with epithelial-mesenchymal transition (EMT), a cellular reprogramming often activated in cancer cells during invasion and metastasis, we investigated whether ALK induces EMT in NSCLC. We performed RNA sequencing analysis on human ALK-rearranged NSCLC cell lines treated with ALK inhibitors or where EML4-ALK was knocked-down by shRNA. We found that EML-ALK regulated several genes related to EMT. In particular, the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2), key regulators of a splicing switch during EMT, were repressed in ALK-rearranged NSCLC cells and their repression was dependent on ALK activity. In keeping with these observations, H2228 and DFCI032 cells displayed a mesenchymal phenotype with almost complete suppression of the epithelial marker, E-cadherin, and a strong expression of the mesenchymal markers, vimentin and N-cadherin. In H2228 and DFCI032 cells, both E-cadherin suppression and vimentin up-regulation were dependent upon EML-ALK kinase activity because treatment with ALK inhibitors (TAE684 and crizotinib) or ALK knock-down reverted the phenotype of H2228 and DFCI032 from mesenchymal to epithelial and decreased their invasive potential. We excluded the involvement in EMT of ALK-rearranged NSCLC of other RTKs, such as EGFR or MET, because their inhibition did not have any effect on E-cadherin and vimentin expression. In ALK-rearranged NSCLC, the regulation of E-cadherin suppression was mainly transcriptional whereas vimentin regulation was post-transcriptional for both cell lines. Overexpression of ESRP1 led to up-regulation of E-cadherin, whereas ESRP1 knock-down impaired the reversion to an epithelial phenotype associated to inhibition of ALK activity. In conclusion, we showed that oncogenic ALK regulates EMT in NSCLC through ESRP repression. These findings could have implications for the biology of ALK-rearranged NSCLC in terms of metastatic potential and resistance to therapy. Citation Format: Claudia Voena, Lydia Varesio, Liye Zhang, Matteo Menotti, Teresa Poggio, Filomena Di Giacomo, Elena Panizza, Cristina Mastini, Mara Compagno, Stefano Monti, Roberto Chiarle. ALK oncogene regulates epithelial-mesenchymal transition (EMT) in ALK-rearranged non-small cell lung carcinoma through repression of the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 131. doi:10.1158/1538-7445.AM2015-131