Publications by authors named "Joan Montero"

27 Publications

  • Page 1 of 1

MCL-1 Inhibition Overcomes Anti-apoptotic Adaptation to Targeted Therapies in B-Cell Precursor Acute Lymphoblastic Leukemia.

Front Cell Dev Biol 2021 9;9:695225. Epub 2021 Sep 9.

Institute for Bioengineering of Catalonia (IBEC), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain.

Multiple targeted therapies are currently explored for pediatric and young adult B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treatment. However, this new armamentarium of therapies faces an old problem: choosing the right treatment for each patient. The lack of predictive biomarkers is particularly worrying for pediatric patients since it impairs the implementation of new treatments in the clinic. In this study, we used the functional assay dynamic BH3 profiling (DBP) to evaluate two new treatments for BCP-ALL that could improve clinical outcome, especially for relapsed patients. We found that the MEK inhibitor trametinib and the multi-target tyrosine kinase inhibitor sunitinib exquisitely increased apoptotic priming in an NRAS-mutant and in a -rearranged cell line presenting a high expression of FLT3, respectively. Following these observations, we sought to study potential adaptations to these treatments. Indeed, we identified with DBP anti-apoptotic changes in the BCL-2 family after treatment, particularly involving MCL-1 - a pro-survival strategy previously observed in adult cancers. To overcome this adaptation, we employed the BH3 mimetic S63845, a specific MCL-1 inhibitor, and evaluated its sequential addition to both kinase inhibitors to overcome resistance. We observed that the metronomic combination of both drugs with S63845 was synergistic and showed an increased efficacy compared to single agents. Similar observations were made in BCP-ALL -rearranged PDX cells in response to sunitinib, showing an analogous DBP profile to the SEM cell line. These findings demonstrate that rational sequences of targeted agents with BH3 mimetics, now extensively explored in clinical trials, may improve treatment effectiveness by overcoming anti-apoptotic adaptations in BCP-ALL.
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http://dx.doi.org/10.3389/fcell.2021.695225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8458912PMC
September 2021

ER+ Breast Cancer Strongly Depends on MCL-1 and BCL-xL Anti-Apoptotic Proteins.

Cells 2021 07 2;10(7). Epub 2021 Jul 2.

Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), 08028 Barcelona, Spain.

Breast cancer is the most frequent type of cancer and the major cause of mortality in women. The rapid development of various therapeutic options has led to the improvement of treatment outcomes; nevertheless, one-third of estrogen receptor (ER)-positive patients relapse due to cancer cell acquired resistance. Here, we use dynamic BH3 profiling (DBP), a functional predictive assay that measures net changes in apoptotic priming, to find new effective treatments for ER+ breast cancer. We observed anti-apoptotic adaptations upon treatment that pointed to metronomic therapeutic combinations to enhance cytotoxicity and avoid resistance. Indeed, we found that the anti-apoptotic proteins BCL-xL and MCL-1 are crucial for ER+ breast cancer cells resistance to therapy, as they exert a dual inhibition of the pro-apoptotic protein BIM and compensate for each other. In addition, we identified the AKT inhibitor ipatasertib and two BH3 mimetics targeting these anti-apoptotic proteins, S63845 and A-1331852, as new potential therapies for this type of cancer. Therefore, we postulate the sequential inhibition of both proteins using BH3 mimetics as a new treatment option for refractory and relapsed ER+ breast cancer tumors.
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http://dx.doi.org/10.3390/cells10071659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8304651PMC
July 2021

Cell Line-Specific Network Models of ER Breast Cancer Identify Potential PI3Kα Inhibitor Resistance Mechanisms and Drug Combinations.

Cancer Res 2021 Sep 13;81(17):4603-4617. Epub 2021 Jul 13.

Eli and Edythe L. Broad Institute of MIT and Harvard, Cambridge, Massachusetts.

Durable control of invasive solid tumors necessitates identifying therapeutic resistance mechanisms and effective drug combinations. In this work, we used a network-based mathematical model to identify sensitivity regulators and drug combinations for the PI3Kα inhibitor alpelisib in estrogen receptor positive (ER) -mutant breast cancer. The model-predicted efficacious combination of alpelisib and BH3 mimetics, for example, MCL1 inhibitors, was experimentally validated in ER breast cancer cell lines. Consistent with the model, FOXO3 downregulation reduced sensitivity to alpelisib, revealing a novel potential resistance mechanism. Cell line-specific sensitivity to combinations of alpelisib and BH3 mimetics depended on which BCL2 family members were highly expressed. On the basis of these results, newly developed cell line-specific network models were able to recapitulate the observed differential response to alpelisib and BH3 mimetics. This approach illustrates how network-based mathematical models can contribute to overcoming the challenge of cancer drug resistance. SIGNIFICANCE: Network-based mathematical models of oncogenic signaling and experimental validation of its predictions can identify resistance mechanisms for targeted therapies, as this study demonstrates for PI3Kα-specific inhibitors in breast cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-21-1208DOI Listing
September 2021

CDK4/6 inhibition reprograms the breast cancer enhancer landscape by stimulating AP-1 transcriptional activity.

Nat Cancer 2021 Jan 9;2(1):34-48. Epub 2020 Nov 9.

Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.

Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) were designed to induce cancer cell cycle arrest. Recent studies have suggested that these agents also exert other effects, influencing cancer cell immunogenicity, apoptotic responses, and differentiation. Using cell-based and mouse models of breast cancer together with clinical specimens, we show that CDK4/6 inhibitors induce remodeling of cancer cell chromatin characterized by widespread enhancer activation, and that this explains many of these effects. The newly activated enhancers include classical super-enhancers that drive luminal differentiation and apoptotic evasion, as well as a set of enhancers overlying endogenous retroviral elements that is enriched for proximity to interferon-driven genes. Mechanistically, CDK4/6 inhibition increases the level of several Activator Protein-1 (AP-1) transcription factor proteins, which are in turn implicated in the activity of many of the new enhancers. Our findings offer insights into CDK4/6 pathway biology and should inform the future development of CDK4/6 inhibitors.
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http://dx.doi.org/10.1038/s43018-020-00135-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8115221PMC
January 2021

PI3Kδ inhibition reshapes follicular lymphoma-immune microenvironment cross talk and unleashes the activity of venetoclax.

Blood Adv 2020 09;4(17):4217-4231

Department of Hematology-Oncology, Institut d'Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS), Barcelona, Spain.

Despite idelalisib approval in relapsed follicular lymphoma (FL), a complete characterization of the immunomodulatory consequences of phosphatidylinositol 3-kinase δ (PI3Kδ) inhibition, biomarkers of response, and potential combinatorial therapies in FL remain to be established. Using ex vivo cocultures of FL patient biopsies and follicular dendritic cells (FDCs) to mimic the germinal center (n = 42), we uncovered that PI3Kδ inhibition interferes with FDC-induced genes related to angiogenesis, extracellular matrix formation, and transendothelial migration in a subset of FL samples, defining an 18-gene signature fingerprint of idelalisib sensitivity. A common hallmark of idelalisib found in all FL cases was its interference with the CD40/CD40L pathway and induced proliferation, together with the downregulation of proteins crucial for B-T-cell synapses, leading to an inefficient cross talk between FL cells and the supportive T-follicular helper cells (TFH). Moreover, idelalisib downmodulates the chemokine CCL22, hampering the recruitment of TFH and immunosupressive T-regulatory cells to the FL niche, leading to a less supportive and tolerogenic immune microenvironment. Finally, using BH3 profiling, we uncovered that FL-FDC and FL-macrophage cocultures augment tumor addiction to BCL-XL and MCL-1 or BFL-1, respectively, limiting the cytotoxic activity of the BCL-2 inhibitor venetoclax. Idelalisib restored FL dependence on BCL-2 and venetoclax activity. In summary, idelalisib exhibits a patient-dependent activity toward angiogenesis and lymphoma dissemination. In all FL cases, idelalisib exerts a general reshaping of the FL immune microenvironment and restores dependence on BCL-2, predisposing FL to cell death, providing a mechanistic rationale for investigating the combination of PI3Kδ inhibitors and venetoclax in clinical trials.
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http://dx.doi.org/10.1182/bloodadvances.2020001584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7479943PMC
September 2020

Sequential combinations of chemotherapeutic agents with BH3 mimetics to treat rhabdomyosarcoma and avoid resistance.

Cell Death Dis 2020 08 15;11(8):634. Epub 2020 Aug 15.

Institute for Bioengineering of Catalonia (IBEC), Barcelona Institute of Science and Technology (BIST), 08028, Barcelona, Spain.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood and adolescence. Refractory/relapsed RMS patients present a bad prognosis that combined with the lack of specific biomarkers impairs the development of new therapies. Here, we utilize dynamic BH3 profiling (DBP), a functional predictive biomarker that measures net changes in mitochondrial apoptotic signaling, to identify anti-apoptotic adaptations upon treatment. We employ this information to guide the use of BH3 mimetics to specifically inhibit BCL-2 pro-survival proteins, defeat resistance and avoid relapse. Indeed, we found that BH3 mimetics that selectively target anti-apoptotic BCL-xL and MCL-1, synergistically enhance the effect of clinically used chemotherapeutic agents vincristine and doxorubicin in RMS cells. We validated this strategy in vivo using a RMS patient-derived xenograft model and observed a reduction in tumor growth with a tendency to stabilization with the sequential combination of vincristine and the MCL-1 inhibitor S63845. We identified the molecular mechanism by which RMS cells acquire resistance to vincristine: an enhanced binding of BID and BAK to MCL-1 after drug exposure, which is suppressed by subsequently adding S63845. Our findings validate the use of DBP as a functional assay to predict treatment effectiveness in RMS and provide a rationale for combining BH3 mimetics with chemotherapeutic agents to avoid tumor resistance, improve treatment efficiency, and decrease undesired secondary effects.
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http://dx.doi.org/10.1038/s41419-020-02887-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7429859PMC
August 2020

A New CDK9 Inhibitor on the Block to Treat Hematologic Malignancies.

Clin Cancer Res 2020 02 16;26(4):761-763. Epub 2019 Dec 16.

Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain.

CDK9-specific inhibition with AZD4573 impairs cancer-promoting gene expression such as and has been proven effective in hematologic malignancies preclinical models. This new clinical candidate should be further explored in the clinic not only as a monotherapy but also in combination with BH3 mimetics to prevent treatment resistance..
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3670DOI Listing
February 2020

Destabilization of NOXA mRNA as a common resistance mechanism to targeted therapies.

Nat Commun 2019 11 14;10(1):5157. Epub 2019 Nov 14.

Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 450 Brookline Ave, Boston, 02115, MA, USA.

Most targeted cancer therapies fail to achieve complete tumor regressions or attain durable remissions. To understand why these treatments fail to induce robust cytotoxic responses despite appropriately targeting oncogenic drivers, here we systematically interrogated the dependence of cancer cells on the BCL-2 family of apoptotic proteins after drug treatment. We observe that multiple targeted therapies, including BRAF or EGFR inhibitors, rapidly deplete the pro-apoptotic factor NOXA, thus creating a dependence on the anti-apoptotic protein MCL-1. This adaptation requires a pathway leading to destabilization of the NOXA mRNA transcript. We find that interruption of this mechanism of anti-apoptotic adaptive resistance dramatically increases cytotoxic responses in cell lines and a murine melanoma model. These results identify NOXA mRNA destabilization/MCL-1 adaptation as a non-genomic mechanism that limits apoptotic responses, suggesting that sequencing of MCL-1 inhibitors with targeted therapies could overcome such widespread and clinically important resistance.
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http://dx.doi.org/10.1038/s41467-019-12477-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856172PMC
November 2019

Pooled Genomic Screens Identify Anti-apoptotic Genes as Targetable Mediators of Chemotherapy Resistance in Ovarian Cancer.

Mol Cancer Res 2019 11 28;17(11):2281-2293. Epub 2019 Aug 28.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.

High-grade serous ovarian cancer (HGSOC) is often sensitive to initial treatment with platinum and taxane combination chemotherapy, but most patients relapse with chemotherapy-resistant disease. To systematically identify genes modulating chemotherapy response, we performed pooled functional genomic screens in HGSOC cell lines treated with cisplatin, paclitaxel, or cisplatin plus paclitaxel. Genes in the intrinsic pathway of apoptosis were among the top candidate resistance genes in both gain-of-function and loss-of-function screens. In an open reading frame overexpression screen, followed by a mini-pool secondary screen, anti-apoptotic genes including (BCL-XL) and (BCL-W) were associated with chemotherapy resistance. In a CRISPR-Cas9 knockout screen, loss of decreased cell survival whereas loss of proapoptotic genes promoted resistance. To dissect the role of individual anti-apoptotic proteins in HGSOC chemotherapy response, we evaluated overexpression or inhibition of BCL-2, BCL-XL, BCL-W, and MCL1 in HGSOC cell lines. Overexpression of anti-apoptotic proteins decreased apoptosis and modestly increased cell viability upon cisplatin or paclitaxel treatment. Conversely, specific inhibitors of BCL-XL, MCL1, or BCL-XL/BCL-2, but not BCL-2 alone, enhanced cell death when combined with cisplatin or paclitaxel. Anti-apoptotic protein inhibitors also sensitized HGSOC cells to the poly (ADP-ribose) polymerase inhibitor olaparib. These unbiased screens highlight anti-apoptotic proteins as mediators of chemotherapy resistance in HGSOC, and support inhibition of BCL-XL and MCL1, alone or combined with chemotherapy or targeted agents, in treatment of primary and recurrent HGSOC. IMPLICATIONS: Anti-apoptotic proteins modulate drug resistance in ovarian cancer, and inhibitors of BCL-XL or MCL1 promote cell death in combination with chemotherapy.
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http://dx.doi.org/10.1158/1541-7786.MCR-18-1243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825578PMC
November 2019

DNA methyltransferase inhibition overcomes diphthamide pathway deficiencies underlying CD123-targeted treatment resistance.

J Clin Invest 2019 11;129(11):5005-5019

Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

The interleukin-3 receptor α subunit, CD123, is expressed in many hematologic malignancies including acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN). Tagraxofusp (SL-401) is a CD123-targeted therapy consisting of interleukin-3 fused to a truncated diphtheria toxin payload. Factors influencing response to tagraxofusp other than CD123 expression are largely unknown. We interrogated tagraxofusp resistance in patients and experimental models and found that it was not associated with CD123 loss. Rather, resistant AML and BPDCN cells frequently acquired deficiencies in the diphthamide synthesis pathway, impairing tagraxofusp's ability to ADP-ribosylate cellular targets. Expression of DPH1, encoding a diphthamide pathway enzyme, was reduced by DNA CpG methylation in resistant cells. Treatment with the DNA methyltransferase inhibitor azacitidine restored DPH1 expression and tagraxofusp sensitivity. We also developed a drug-dependent ADP-ribosylation assay in primary cells that correlated with tagraxofusp activity and may represent an additional novel biomarker. As predicted by these results and our observation that resistance also increased mitochondrial apoptotic priming, we found that the combination of tagraxofusp and azacitidine was effective in patient-derived xenografts treated in vivo. These data have important implications for clinical use of tagraxofusp and led to a phase 1 study combining tagraxofusp and azacitidine in myeloid malignancies.
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http://dx.doi.org/10.1172/JCI128571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6819120PMC
November 2019

Dual inhibition of MDM2 and MDM4 in virus-positive Merkel cell carcinoma enhances the p53 response.

Proc Natl Acad Sci U S A 2019 01 31;116(3):1027-1032. Epub 2018 Dec 31.

Program in Virology, Graduate School of Arts and Sciences, Harvard University, Cambridge, MA 02138;

Merkel cell polyomavirus (MCV) contributes to approximately 80% of all Merkel cell carcinomas (MCCs), a highly aggressive neuroendocrine carcinoma of the skin. MCV-positive MCC expresses small T antigen (ST) and a truncated form of large T antigen (LT) and usually contains wild-type p53 (TP53) and RB (RB1). In contrast, virus-negative MCC contains inactivating mutations in TP53 and RB1. While the MCV-truncated LT can bind and inhibit RB, it does not bind p53. We report here that MCV LT binds to RB, leading to increased levels of ARF, an inhibitor of MDM2, and activation of p53. However, coexpression of ST reduced p53 activation. MCV ST recruits the MYC homologue MYCL (L-Myc) to the EP400 chromatin remodeler complex and transactivates specific target genes. We observed that depletion of EP400 in MCV-positive MCC cell lines led to increased p53 target gene expression. We suspected that the MCV ST-MYCL-EP400 complex could functionally inactivate p53, but the underlying mechanism was not known. Integrated ChIP and RNA-sequencing analysis following EP400 depletion identified MDM2 as well as CK1α, an activator of MDM4, as target genes of the ST-MYCL-EP400 complex. In addition, MCV-positive MCC cells expressed high levels of MDM4. Combining MDM2 inhibitors with lenalidomide targeting CK1α or an MDM4 inhibitor caused synergistic activation of p53, leading to an apoptotic response in MCV-positive MCC cells and MCC-derived xenografts in mice. These results support dual targeting of MDM2 and MDM4 in virus-positive MCC and other p53 wild-type tumors.
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http://dx.doi.org/10.1073/pnas.1818798116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338866PMC
January 2019

Targeted apoptosis of myofibroblasts with the BH3 mimetic ABT-263 reverses established fibrosis.

Sci Transl Med 2017 Dec;9(420)

Fibrosis Research Center and Center for Immunology and Inflammatory Diseases, Division of Pulmonary and Critical Care Medicine, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.

Persistent myofibroblast activation distinguishes pathological fibrosis from physiological wound healing, suggesting that therapies selectively inducing myofibroblast apoptosis could prevent progression and potentially reverse established fibrosis in diseases such as scleroderma, a heterogeneous autoimmune disease characterized by multiorgan fibrosis. We demonstrate that fibroblast-to-myofibroblast differentiation driven by matrix stiffness increases the mitochondrial priming (proximity to the apoptotic threshold) of these activated cells. Mitochondria in activated myofibroblasts, but not quiescent fibroblasts, are primed by death signals such as the proapoptotic BH3-only protein BIM, which creates a requirement for tonic expression of the antiapoptotic protein BCL-X to sequester BIM and ensure myofibroblast survival. Myofibroblasts become particularly susceptible to apoptosis induced by "BH3 mimetic" drugs inhibiting BCL-X such as ABT-263. ABT-263 displaces BCL-X binding to BIM, allowing BIM to activate apoptosis on stiffness-primed myofibroblasts. Therapeutic blockade of BCL-X with ABT-263 (navitoclax) effectively treats established fibrosis in a mouse model of scleroderma dermal fibrosis by inducing myofibroblast apoptosis. Using a BH3 profiling assay to assess mitochondrial priming in dermal fibroblasts derived from patients with scleroderma, we demonstrate that the extent of apoptosis induced by BH3 mimetic drugs correlates with the extent of their mitochondrial priming, indicating that BH3 profiling could predict apoptotic responses of fibroblasts to BH3 mimetic drugs in patients with scleroderma. Together, our findings elucidate the potential efficacy of targeting myofibroblast antiapoptotic proteins with BH3 mimetic drugs in scleroderma and other fibrotic diseases.
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http://dx.doi.org/10.1126/scitranslmed.aal3765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8520471PMC
December 2017

Why do BCL-2 inhibitors work and where should we use them in the clinic?

Cell Death Differ 2018 01 27;25(1):56-64. Epub 2017 Oct 27.

Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA.

Intrinsic apoptosis is controlled by the BCL-2 family of proteins but the complexity of intra-family interactions makes it challenging to predict cell fate via standard molecular biology techniques. We discuss BCL-2 family regulation and how to determine cells' readiness for apoptosis and anti-apoptotic dependence. Cancer cells often adopt anti-apoptotic defense mechanisms in response to oncogenic stress or anti-cancer therapy. However, by determining their anti-apoptotic addiction, we can use novel BH3 mimetics to overwhelm this apoptotic blockade. We outline the development and uses of these unique anti-apoptotic inhibitors and how to possibly combine them with other anti-cancer agents using dynamic BH3 profiling (DBP) to improve personalized cancer treatment.
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http://dx.doi.org/10.1038/cdd.2017.183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5729538PMC
January 2018

The 2-oxoglutarate carrier promotes liver cancer by sustaining mitochondrial GSH despite cholesterol loading.

Redox Biol 2018 04 14;14:164-177. Epub 2017 Sep 14.

Department of Cell Death and Proliferation, Instituto de Investigaciones Biomédicas de Barcelona, Consejo Superior de Investigaciones Científicas, 08036 Barcelona, Spain; Liver Unit and Hospital Clínic i Provincial, IDIBAPS, and Centro de Investigación Biomédica en Red (CIBERehd), Spain; Southern California Research Center for ALPD and Cirrhosis, Los Angeles, CA, USA; University of Southern California Research Center for Liver Diseases, Keck School of Medicine, USC, Los Angeles, CA, USA. Electronic address:

Cancer cells exhibit mitochondrial cholesterol (mt-cholesterol) accumulation, which contributes to cell death resistance by antagonizing mitochondrial outer membrane (MOM) permeabilization. Hepatocellular mt-cholesterol loading, however, promotes steatohepatitis, an advanced stage of chronic liver disease that precedes hepatocellular carcinoma (HCC), by depleting mitochondrial GSH (mGSH) due to a cholesterol-mediated impairment in mGSH transport. Whether and how HCC cells overcome the restriction of mGSH transport imposed by mt-cholesterol loading to support mGSH uptake remains unknown. Although the transport of mGSH is not fully understood, SLC25A10 (dicarboxylate carrier, DIC) and SLC25A11 (2-oxoglutarate carrier, OGC) have been involved in mGSH transport, and therefore we examined their expression and role in HCC. Unexpectedly, HCC cells and liver explants from patients with HCC exhibit divergent expression of these mitochondrial carriers, with selective OGC upregulation, which contributes to mGSH maintenance. OGC but not DIC downregulation by siRNA depleted mGSH levels and sensitized HCC cells to hypoxia-induced ROS generation and cell death as well as impaired cell growth in three-dimensional multicellular HCC spheroids, effects that were reversible upon mGSH replenishment by GSH ethyl ester, a membrane permeable GSH precursor. We also show that OGC regulates mitochondrial respiration and glycolysis. Moreover, OGC silencing promoted hypoxia-induced cardiolipin peroxidation, which reversed the inhibition of cholesterol on the permeabilization of MOM-like liposomes induced by Bax or Bak. Genetic OGC knockdown reduced the ability of tumor-initiating stem-like cells to induce liver cancer. These findings underscore the selective overexpression of OGC as an adaptive mechanism of HCC to provide adequate mGSH levels in the face of mt-cholesterol loading and suggest that OGC may be a novel therapeutic target for HCC treatment.
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http://dx.doi.org/10.1016/j.redox.2017.08.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5609874PMC
April 2018

Blastic Plasmacytoid Dendritic Cell Neoplasm Is Dependent on BCL2 and Sensitive to Venetoclax.

Cancer Discov 2017 02 16;7(2):156-164. Epub 2016 Dec 16.

Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive hematologic malignancy with dismal outcomes for which no standard therapy exists. We found that primary BPDCN cells were dependent on the antiapoptotic protein BCL2 and were uniformly sensitive to the BCL2 inhibitor venetoclax, as measured by direct cytotoxicity, apoptosis assays, and dynamic BH3 profiling. Animals bearing BPDCN patient-derived xenografts had disease responses and improved survival after venetoclax treatment in vivo Finally, we report on 2 patients with relapsed/refractory BPDCN who received venetoclax off-label and experienced significant disease responses. We propose that venetoclax or other BCL2 inhibitors undergo expedited clinical evaluation in BPDCN, alone or in combination with other therapies. In addition, these data illustrate an example of precision medicine to predict treatment response using ex vivo functional assessment of primary tumor tissue, without requiring a genetic biomarker.

Significance: Therapy for BPDCN is inadequate, and survival in patients with the disease is poor. We used primary tumor cell functional profiling to predict BCL2 antagonist sensitivity as a common feature of BPDCN, and demonstrated in vivo clinical activity of venetoclax in patient-derived xenografts and in 2 patients with relapsed chemotherapy-refractory disease. Cancer Discov; 7(2); 156-64. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 115.
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http://dx.doi.org/10.1158/2159-8290.CD-16-0999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5296248PMC
February 2017

Dynamic BH3 profiling-poking cancer cells with a stick.

Mol Cell Oncol 2016 May 10;3(3):e1040144. Epub 2016 Mar 10.

Dana-Farber Cancer Institute, Harvard Medical School , Boston, MA, USA.

The vast majority of efforts in precision medicine for cancer try to link static genetic information to tumor biology and from there predict clinical response. Dynamic BH3 profiling offers an alternative functional approach by measuring death signaling induced by specific drugs in tumors from patients ex vivo to predict clinical response.
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http://dx.doi.org/10.1080/23723556.2015.1040144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4909438PMC
May 2016

Complementary dynamic BH3 profiles predict co-operativity between the multi-kinase inhibitor TG02 and the BH3 mimetic ABT-199 in acute myeloid leukaemia cells.

Oncotarget 2017 Mar;8(10):16220-16232

Clinical Haematology, Nottingham University Hospitals, Nottingham, UK.

Direct co-operation between sensitiser molecules BAD and NOXA in mediating apoptosis suggests that therapeutic agents which sensitise to BAD may complement agents which sensitise to NOXA. Dynamic BH3 profiling is a novel methodology that we have applied to the measurement of complementarity between sensitiser BH3 peptide mimetics and therapeutic agents. Using dynamic BH3 profiling, we show that the agent TG02, which downregulates MCL-1, sensitises to the BCL-2-inhibitory BAD-BH3 peptide, whereas the BCL-2 antagonist ABT-199 sensitises to MCL-1 inhibitory NOXA-BH3 peptide in acute myeloid leukaemia (AML) cells. At the concentrations used, the peptides did not trigger mitochondrial outer membrane permeabilisation in their own right, but primed cells to release Cytochrome C in the presence of an appropriate trigger of a complementary pathway. In KG-1a cells TG02 and ABT-199 synergised to induce apoptosis. In heterogeneous AML patient samples we noted a range of sensitivities to the two agents. Although some individual samples markedly favoured one agent or the other, in the group as a whole the combination of TG02 + ABT-199 was significantly more cytotoxic than either agent individually. We conclude that dynamic NOXA and BAD BH3 profiling is a sensitive methodology for investigating molecular pathways of drug action and complementary mechanisms of chemoresponsiveness.
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http://dx.doi.org/10.18632/oncotarget.8742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5369958PMC
March 2017

The Public Repository of Xenografts Enables Discovery and Randomized Phase II-like Trials in Mice.

Cancer Cell 2016 04;29(4):574-586

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, 450 Brookline Avenue, Dana 510B, MA 02215, USA.

More than 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publicly available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment, called the Public Repository of Xenografts (PRoXe). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional, and proteomic biomarkers in both treatment-naive and relapsed/refractory disease.
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http://dx.doi.org/10.1016/j.ccell.2016.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5177991PMC
April 2016

iBH3: simple, fixable BH3 profiling to determine apoptotic priming in primary tissue by flow cytometry.

Biol Chem 2016 07;397(7):671-8

Dysregulation of the mitochondrial pathway of apoptosis, controlled by the BCL-2 family of proteins, leads to disease states including cancer. Rapid analysis of a cell's dependency on the BCL-2 family of proteins is hindered by the complex interactions of more than a dozen proteins. Transcript or even protein levels are therefore generally insufficient to predict a cell's response to perturbations like chemotherapy. Previously, we developed the JC-1 BH3 method to provide a same day functional assay to assess a cell's propensity to undergo apoptosis and demonstrated its utility in predicting response to chemotherapy. We have now improved upon these methods to create a robust assay amenable to high throughput platforms using cytochrome c retention in formaldehyde fixed cells to remove the time sensitivity of JC-1 potential measurements. BH3 profiling by intracellular staining (iBH3) is suitable for 96- and 384-well formats, and can be used to directly screen candidate BH3-mimetic compounds for activity. When used as the final component of dynamic BH3 profiling (DBP), which uses a drug pretreatment prior to iBH3 to assess the change in profile due to treatment, it can predict the response of cells to chemotherapy days before they show signs of death.
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http://dx.doi.org/10.1515/hsz-2016-0107DOI Listing
July 2016

Activity of the Type II JAK2 Inhibitor CHZ868 in B Cell Acute Lymphoblastic Leukemia.

Cancer Cell 2015 Jul;28(1):29-41

Novartis Institutes for Biomedical Research, 4002 Basel, Switzerland.

A variety of cancers depend on JAK2 signaling, including the high-risk subset of B cell acute lymphoblastic leukemias (B-ALLs) with CRLF2 rearrangements. Type I JAK2 inhibitors induce paradoxical JAK2 hyperphosphorylation in these leukemias and have limited activity. To improve the efficacy of JAK2 inhibition in B-ALL, we developed the type II inhibitor CHZ868, which stabilizes JAK2 in an inactive conformation. CHZ868 potently suppressed the growth of CRLF2-rearranged human B-ALL cells, abrogated JAK2 signaling, and improved survival in mice with human or murine B-ALL. CHZ868 and dexamethasone synergistically induced apoptosis in JAK2-dependent B-ALLs and further improved in vivo survival compared to CHZ868 alone. These data support the testing of type II JAK2 inhibition in patients with JAK2-dependent leukemias and other disorders.
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http://dx.doi.org/10.1016/j.ccell.2015.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505625PMC
July 2015

Drug-induced death signaling strategy rapidly predicts cancer response to chemotherapy.

Cell 2015 Feb;160(5):977-989

Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA. Electronic address:

There is a lack of effective predictive biomarkers to precisely assign optimal therapy to cancer patients. While most efforts are directed at inferring drug response phenotype based on genotype, there is very focused and useful phenotypic information to be gained from directly perturbing the patient's living cancer cell with the drug(s) in question. To satisfy this unmet need, we developed the Dynamic BH3 Profiling technique to measure early changes in net pro-apoptotic signaling at the mitochondrion ("priming") induced by chemotherapeutic agents in cancer cells, not requiring prolonged ex vivo culture. We find in cell line and clinical experiments that early drug-induced death signaling measured by Dynamic BH3 Profiling predicts chemotherapy response across many cancer types and many agents, including combinations of chemotherapies. We propose that Dynamic BH3 Profiling can be used as a broadly applicable predictive biomarker to predict cytotoxic response of cancers to chemotherapeutics in vivo.
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http://dx.doi.org/10.1016/j.cell.2015.01.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4391197PMC
February 2015

BID preferentially activates BAK while BIM preferentially activates BAX, affecting chemotherapy response.

Mol Cell 2013 Sep;51(6):751-65

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Harvard Medical School, Boston, MA 02115, USA.

Apoptosis is a highly regulated form of cell death that controls normal homeostasis as well as the antitumor activity of many chemotherapeutic agents. Commitment to death via the mitochondrial apoptotic pathway requires activation of the mitochondrial pore-forming proteins BAK or BAX. Activation can be effected by the activator BH3-only proteins BID or BIM, which have been considered to be functionally redundant in this role. Herein, we show that significant activation preferences exist between these proteins: BID preferentially activates BAK while BIM preferentially activates BAX. Furthermore, we find that cells lacking BAK are relatively resistant to agents that require BID activation for maximal induction of apoptosis, including topoisomerase inhibitors and TRAIL. Consequently, patients with tumors that harbor a loss of BAK1 exhibit an inferior response to topoisomerase inhibitor treatment in the clinic. Therefore, BID and BIM have nonoverlapping roles in the induction of apoptosis via BAK and BAX, affecting chemotherapy response.
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http://dx.doi.org/10.1016/j.molcel.2013.08.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4164233PMC
September 2013

KPT-330 inhibitor of CRM1 (XPO1)-mediated nuclear export has selective anti-leukaemic activity in preclinical models of T-cell acute lymphoblastic leukaemia and acute myeloid leukaemia.

Br J Haematol 2013 Apr 4;161(1):117-27. Epub 2013 Feb 4.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.

This study explored the anti-leukaemic efficacy of novel irreversible inhibitors of the major nuclear export receptor, chromosome region maintenance 1 (CRM1, also termed XPO1). We found that these novel CRM1 antagonists, termed SINE (Selective Inhibitors of Nuclear Export), induced rapid apoptosis at low nanomolar concentrations in a panel of 14 human T-cell acute lymphoblastic leukaemia (T-ALL) cell lines representing different molecular subtypes of the disease. To assess in vivo anti-leukaemia cell activity, we engrafted immunodeficient mice intravenously with the human T-ALL MOLT-4 cells, which harbour activating mutations of NOTCH1 and NRAS as well as loss of function of the CDKN2A, PTEN and TP53 tumour suppressors and express a high level of oncogenic transcription factor TAL1. Importantly, we examined the in vivo anti-leukaemic efficacy of the clinical SINE compound KPT-330 against T-ALL and acute myeloid leukaemia (AML) cells. These studies demonstrated striking in vivo activity of KPT-330 against T-ALL and AML cells, with little toxicity to normal murine haematopoietic cells. Taken together, our results show that SINE CRM1 antagonists represent promising 'first-in-class' drugs with a novel mechanism of action and wide therapeutic index, and imply that drugs of this class show promise for the targeted therapy of T-ALL and AML.
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http://dx.doi.org/10.1111/bjh.12231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3980736PMC
April 2013

Reactivation of ERK signaling causes resistance to EGFR kinase inhibitors.

Cancer Discov 2012 Oct 7;2(10):934-47. Epub 2012 Sep 7.

Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.

The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors is limited by the development of drug resistance. The irreversible EGFR kinase inhibitor WZ4002 is effective against the most common mechanism of drug resistance mediated by the EGFR T790M mutation. Here, we show, in multiple complementary models, that resistance to WZ4002 develops through aberrant activation of extracellular signal-regulated kinase (ERK) signaling caused by either an amplification of mitogen-activated protein kinase 1 (MAPK1) or by downregulation of negative regulators of ERK signaling. Inhibition of MAP-ERK kinase (MEK) or ERK restores sensitivity to WZ4002 and prevents the emergence of drug resistance. We further identify MAPK1 amplification in an erlotinib-resistant EGFR-mutant non-small cell lung carcinoma patient. In addition, the WZ4002-resistant MAPK1-amplified cells also show an increase both in EGFR internalization and a decrease in sensitivity to cytotoxic chemotherapy. Our findings provide insights into mechanisms of drug resistance to EGFR kinase inhibitors and highlight rational combination therapies that should be evaluated in clinical trials.
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http://dx.doi.org/10.1158/2159-8290.CD-12-0103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3477553PMC
October 2012

Cholesterol and peroxidized cardiolipin in mitochondrial membrane properties, permeabilization and cell death.

Biochim Biophys Acta 2010 Jun-Jul;1797(6-7):1217-24. Epub 2010 Feb 11.

Liver Unit and Centro de Investigaciones Biomédicas Esther Koplowitz, IMDiM, Hospital Clínic i Provincial and CIBEREHD, IDIBAPS, and Department of Cell Death and Proliferation, Instituto Investigaciones Biomédicas de Barcelona, Consejo Superior de Investigaciones Científicas, Barcelona, Spain.

Mitochondria are known to actively regulate cell death with the final phenotype of demise being determined by the metabolic and energetic status of the cell. Mitochondrial membrane permeabilization (MMP) is a critical event in cell death, as it regulates the degree of mitochondrial dysfunction and the release of intermembrane proteins that function in the activation and assembly of caspases. In addition to the crucial role of proapoptotic members of the Bcl-2 family, the lipid composition of the mitochondrial membranes is increasingly recognized to modulate MMP and hence cell death. The unphysiological accumulation of cholesterol in mitochondrial membranes regulates their physical properties, facilitating or impairing MMP during Bax and death ligand-induced cell death depending on the level of mitochondrial GSH (mGSH), which in turn regulates the oxidation status of cardiolipin. Cholesterol-mediated mGSH depletion stimulates TNF-induced reactive oxygen species and subsequent cardiolipin peroxidation, which destabilizes the lipid bilayer and potentiates Bax-induced membrane permeabilization. These data suggest that the balance of mitochondrial cholesterol to peroxidized cardiolipin regulates mitochondrial membrane properties and permeabilization, emerging as a rheostat in cell death.
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http://dx.doi.org/10.1016/j.bbabio.2010.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2889134PMC
January 2011

Mitochondrial cholesterol contributes to chemotherapy resistance in hepatocellular carcinoma.

Cancer Res 2008 Jul;68(13):5246-56

Liver Unit and Centro de Investigaciones Biomédicas Esther Koplowitz, IMDiM, Hospital Clínic i Provincial, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain.

Cholesterol metabolism is deregulated in carcinogenesis, and cancer cells exhibit enhanced mitochondrial cholesterol content whose role in cell death susceptibility and cancer therapy has not been investigated. Here, we describe that mitochondria from rat or human hepatocellular carcinoma (HC) cells (HCC) or primary tumors from patients with HC exhibit increased mitochondrial cholesterol levels. HCC sensitivity to chemotherapy acting via mitochondria is enhanced upon cholesterol depletion by inhibition of hydroxymethylglutaryl-CoA reductase or squalene synthase (SS), which catalyzes the first committed step in cholesterol biosynthesis. HCC transfection with siRNA targeting the steroidogenic acute regulatory protein StAR, a mitochondrial cholesterol-transporting polypeptide which is overexpressed in HCC compared with rat and human liver, sensitized HCC to chemotherapy. Isolated mitochondria from HCC with increased cholesterol levels were resistant to mitochondrial membrane permeabilization and release of cytochrome c or Smac/DIABLO in response to various stimuli including active Bax. Similar behavior was observed in cholesterol-enriched mitochondria or liposomes and reversed by restoring mitochondrial membrane order or cholesterol extraction. Moreover, atorvastatin or the SS inhibitor YM-53601 potentiated doxorubicin-mediated HCC growth arrest and cell death in vivo. Thus, mitochondrial cholesterol contributes to chemotherapy resistance by increasing membrane order, emerging as a novel therapeutic niche in cancer therapy.
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http://dx.doi.org/10.1158/0008-5472.CAN-07-6161DOI Listing
July 2008
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