Search

Supplementary Tables - PDF file 148K, Table S1. Characteristics of HER2-negative breast cancers, separated by HR status, TCGA Table S2. Characteristics of triple-negative breast cancers with residual disease post neoadjuvant chemotherapy and univariate Table S3. Bimodal analysis of metabolism-related genes ranked by bimodality index (BI)

Supplementary Figures - PDF file 440K, Figure S1. Figure S1. Validation the IHC protocol for LDHB using FFPE MDAMB231 cells with stable knockdown of LDHA or LDHB. Reduced staining for LDHB was detected only in the LDHB knockdown line. Control cells were infected with a non-silencing shRNA. Figure S2. LDHB, when present, substantially contributed to the total LDH activity in cell lines. Figure S3. A, verification of LDHA or LDHB knockdown by LDH activity in stable isogenic cell lines, MDAMB231 and HCC1937. Figure S4. LDHB association with PAM50 intrinsic subtype in primary breast cancers. A, LDHB mRNA expression separated by intrinsic subtype: Perou GSE10893 (8) Figure S5. High LDHB was associated with triple-negative breast cancer. LDHB mRNA expression of HER2 (by IHC/FISH)-negative disease separated by clinical classification Figure S6. LDHB mRNA expression within PAM50 intrinsic subtypes for HR-positive/HER2-negative breast cancers. Figure S7. LDHB mRNA expression within PAM50 intrinsic subtypes for triple-negative breast cancers Figure S8. LDHB mRNA expression was highly associated with cell cycle proliferation marker, CCNB1, for basal-like cancers within the triple-negative group. Figure S9. Association between mRNA expression and copy number for LDHB for the breast invasive carcinoma cohort separated by (A) basal and (B) luminal A/B subtypes for TCGA "manuscript samples" within the cBio Cancer Genomics Portal

Supplementary Data - PDF file 89K, Supplementary Methods Tissue microarray (TMA) of triple-negative breast cancers

Purpose: Although breast cancers are known to be molecularly heterogeneous, their metabolic phenotype is less well-understood and may predict response to chemotherapy. This study aimed to evaluate metabolic genes as individual predictive biomarkers in breast cancer.Experimental Design: mRNA microarray data from breast cancer cell lines were used to identify bimodal genes—those with highest potential for robust high/low classification in clinical assays. Metabolic function was evaluated in vitro for the highest scoring metabolic gene, lactate dehydrogenase B (LDHB). Its expression was associated with neoadjuvant chemotherapy response and relapse within clinical and PAM50-derived subtypes.Results: LDHB was highly expressed in cell lines with glycolytic, basal-like phenotypes. Stable knockdown of LDHB in cell lines reduced glycolytic dependence, linking LDHB expression directly to metabolic function. Using patient datasets, LDHB was highly expressed in basal-like cancers and could predict basal-like subtype within clinical groups [OR = 21 for hormone receptor (HR)-positive/HER2-negative; OR = 10 for triple-negative]. Furthermore, high LDHB predicted pathologic complete response (pCR) to neoadjuvant chemotherapy for both HR-positive/HER2-negative (OR = 4.1, P < 0.001) and triple-negative (OR = 3.0, P = 0.003) cancers. For triple-negative tumors without pCR, high LDHB posttreatment also identified proliferative tumors with increased risk of recurrence (HR = 2.2, P = 0.006).Conclusions: Expression of LDHB predicted response to neoadjuvant chemotherapy within clinical subtypes independently of standard prognostic markers and PAM50 subtyping. These observations support prospective clinical evaluation of LDHB as a predictive marker of response for patients with breast cancer receiving neoadjuvant chemotherapy. Clin Cancer Res; 19(13); 3703–13. ©2013 AACR.

The type 2 cytokines IL-4 and IL-13, which share use of an IL-4 receptor alpha chain and its nuclear induction of the transcription factor STAT6, are crucial in elicitation and maintenance of allergic conditions that include asthma. Prior work has shown a physical and functional association of STAT6 with PARP14, an ADP-ribosyl monotransferase. Moreover, elimination of all PARP14 expression by gene targeting led to altered recall antibody responses and attenuation of ovalbumin-specific allergic lung inflammation with no apparent health issues for mice lacking this protein. However, an unanswered question is whether or not inhibition of the catalytic function has any biological consequence since PARP14 has multiple functional domains apart from the portion that catalyzes ADP-ribosylation. As reported separately, iterative structural analyses and medicinal chemistry fostered the generation of a compound, RBN2759, that is highly selective in its inhibition of PARP14 with negligible impact on other members of the PARP gene family. We show here that administration of this compound to mice previously sensitized to the allergenAlternaria alternataachieved biochemically active levels and altered physiological responses to the antigen. These results show for the first time that in vivo administration of a specific inhibitor of the ADP-ribosyltransferase activity encoded by PARP14 is sufficient to alter biological responses. Specifically, the orally absorbable pharmaceutical compound decreased allergen-induced mucus, blunted the induced increases in circulating IgE, and prevented suppression of IgG2a. We conclude that the catalytic activity can contribute to pathogenesis in allergic processes and propose that other biological endpoints that depend on ADP-ribosylation by PARP14 can be targeted using selective inhibition.

Targeting cytosolic nucleic acid sensing pathways to activate the Type I interferon (IFN) response is an emerging therapeutic strategy being explored in oncology. The PARP family consists of seventeen enzymes that regulate fundamental biological processes including response to cellular stress. PARP7 (TIPARP) is a stress-induced mono-ART that catalyzes the transfer of a single unit of ADP-ribose onto substrates (MARylation) to regulate their function and plays a role in suppressing the Type I IFN response in tumor cells (Gozgit 2021 Cancer Cell). RBN-2397 is the first potent and selective small molecule inhibitor of PARP7 catalytic function. To investigate the cell autonomous effects of PARP7 inhibition, we performed a cell line screen to identify PARP7 dependent cancer cell lines. We found that treatment of a subset of lines across several cancers led to a robust decrease in cell viability. Additionally, dosing of tumor bearing mice led to complete regressions in NCI-H1373 lung cancer xenografts. To investigate the mechanism of action (MOA) leading to decreased cell viability, we treated NCI-H1373 cells with RBN-2397 and found accumulation of cells in the G0/G1 phase of the cell cycle indicative of a cell cycle arrest. This arrest in NCI-H1373 cells was associated with the induction of senescence and increased mRNA expression of senescence associated secretory phenotype (SASP) genes. To evaluate the in vivo MOA, we performed an NCI-H1373 xenograft study and collected tumors after 7 days of RBN-2397 treatment. PARP7 inhibition led to decreased expression of Ki67, and increased expression of P21 and cleaved caspase-3, suggesting decreased proliferation and increased apoptosis. Increased expression of SASP genes was also observed in RBN-2397 treated tumors. Finally, we investigated transcriptional changes after RBN-2397 treatment by RNA sequencing. In addition to the effects observed in Type I IFN signaling, we also observed differential expression of genes associated with other pathways including autophagy and energy metabolism. Further evaluation of key autophagy proteins revealed that RBN-2397 affects autophagy flux and leads to a decrease in the oxygen consumption rate of cells and reduced ATP production from the mitochondria, suggesting that a change in energy metabolism may be related to the tumor intrinsic effect of RBN-2397. In summary, we show treatment of cancer cells with RBN-2397 not only leads to activation of tumor cell IFN signaling, but also causes G1 arrest and senescence, and changes in cancer cell autophagy and energy metabolism. In vivo, RBN-2397 treatment leads to complete tumor regressions in xenografts accompanied by decreased proliferation and increased apoptosis of tumor cells. RBN-2397 is currently being evaluated in the clinic as single agent in selected cancer types (NCT04053673) and in combination with anti-PD-1 therapies. Citation Format: Jennifer R. Molina, Joseph M. Gozgit, Melissa M. Vasbinder, Ryan P. Abo, Kaiko kunii, Kristy G. Kuplast-Barr, Bin Gui, Sunaina P. Nayak, Elena Minissale, Kerren K. Swinger, Tim J. Wigle, Alvin Z. Lu, Danielle J. Blackwell, Christina R. Majer, Yue Ren, Ellen Bamberg, Mario Niepel, Jan-Rung Mo, William D. Church, Ahmed S. Mady, Jeff Song, Zacharenia A. Varsamis, Luke Utley, Patricia E. Rao, Timoty J. Mitchison, Kevin W. Kuntz, Victoria M. Richon, Kristen McEachern, Heike Keilhack. PARP7 inhibitor RBN-2397 increases tumoral IFN signaling leading to various tumor cell intrinsic effects and tumor regressions in mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2154.

PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function. Here, we identify PARP7 as a negative regulator of nucleic acid sensing in tumor cells. Inhibition of PARP7 restores type I interferon (IFN) signaling responses to nucleic acids in tumor models. Restored signaling can directly inhibit cell proliferation and activate the immune system, both of which contribute to tumor regression. Oral dosing of the PARP7 small-molecule inhibitor, RBN-2397, results in complete tumor regression in a lung cancer xenograft and induces tumor-specific adaptive immune memory in an immunocompetent mouse cancer model, dependent on inducing type I IFN signaling in tumor cells. PARP7 is a therapeutic target whose inhibition induces both cancer cell-autonomous and immune stimulatory effects via enhanced IFN signaling. These data support the targeting of a monoPARP in cancer and introduce a potent and selective PARP7 inhibitor to enter clinical development.

Summary PARP14 has been implicated by genetic knockout studies to promote protumor macrophage polarization and suppress the antitumor inflammatory response due to its role in modulating interleukin-4 (IL-4) and interferon-γ signaling pathways. Here, we describe structure-based design efforts leading to the discovery of a potent and highly selective PARP14 chemical probe. RBN012759 inhibits PARP14 with a biochemical half-maximal inhibitory concentration of 0.003 μM, exhibits >300-fold selectivity over all PARP family members, and its profile enables further study of PARP14 biology and disease association both in vitro and in vivo. Inhibition of PARP14 with RBN012759 reverses IL-4-driven protumor gene expression in macrophages and induces an inflammatory mRNA signature similar to that induced by immune checkpoint inhibitor therapy in primary human tumor explants. These data support an immune suppressive role of PARP14 in tumors and suggest potential utility of PARP14 inhibitors in the treatment of cancer.

Therapeutic agents targeting metabolism in the tumor microenvironment have been of increasing interest in recent years, however, the complexities of the interplay between tumor, stromal and immune cell interactions add complexity to these therapeutic approaches. We have previously disclosed the development of the GLS1 inhibitor, IPN60090, which is currently progressing through Phase 1 studies (NCT03894540) in multiple indication-specific biomarker-positive patient populations. In order to fully appreciate the opportunity to enhance IPN60090 activity in patients, we explored the impact of GLS1 inhibition on the activity of the immune system. Glutamine metabolism has been shown to play important and varied roles within the immune compartment including, but not limited to, roles in T-cell activation, T-cell effector functions and progression of exhaustion phenotypes. During T-cell activation, glutamine is utilized to drive activated T-cells towards a glycolytic phenotype. Eventually, activated T-cells exhaust and shift away from glycolysis towards fatty acid oxidation. Interestingly, it has been reported that the interaction of PD1 with PD-L1 blocks glutamine import and decreases glycolysis. Given these data, we hypothesized that inhibition of GLS1 with IPN60090 might enhance checkpoint blockade by increasing levels of glutamine in the tumor microenvironment, thus enhancing anti-tumor immune responses. In ex vivo culture systems, we show that IPN60090-mediated GLS1 inhibition increases the glycolytic activity of CD4+ and CD8+ T-cells, suggesting that GLS1 inhibition allows cells to maintain an energetically favorable phenotype. Furthermore, we show that IPN60090 enhances checkpoint blockade through cooperation with αPD1 therapy in two syngeneic mouse models which do not harbor predictive biomarkers of response to IPN60090 and which are refractory to checkpoint blockade. The observed synergy is due in part to IPN60090-dependent depletion of regulatory T-cells (Treg) and a concurrent increase in the CD8+ T-cell to Treg ratio in the tumor microenvironment. These data suggest that IPN60090 may show clinical benefit by enhancing immune response in the context of checkpoint blockade and have served as the justification for phase 1b trials in combination with Pembrolizumab which will enroll in 2021. Citation Format: Erika Suzuki, Jennifer Molina, Nakia D. Spencer, Christopher A. Bristow, Angela L. Harris, Ningping Feng, Mikhila Mahendra, Sonal Gera, Michael J. Soth, Kang Le, Timothy A. Yap, Giulio Draetta, Philip Jones, Timothy P. Heffernan, Jeffrey J. Kovacs. The GLS1 inhibitor IPN60090 enhances antitumor immune response through metabolic reprogramming of T cells and impacts on the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2338.

Targeting cytosolic nucleic acid sensing pathways and the Type I interferon (IFN) response is an emerging therapeutic strategy being explored in oncology. The PARP family consists of seventeen enzymes that regulate fundamental biological processes including response to cellular stress. In contrast to PARP1, PARP7 (TIPARP) is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates (MARylation) to change their function and plays a role in suppressing the Type I IFN response. RBN-2397 selectively inhibits PARP7 compared to the approved PARP1 inhibitors and demonstrates > 50-fold selectivity for inhibition of PARP7 over all PARP family members as measured by biochemical assays. The inhibition of PARP1-mediated ADP-ribosylation has been well-characterized for several PARP1 inhibitors using a cellular hydrogen peroxide-induced PARylation assay. Here, we show that RBN-2397 inhibits PARP7-dependent MARylation with an IC50 of 2 nM exhibiting a 300-fold window over PARP1-driven PARylation. Using the mouse CT26 cell line, we showed that RBN-2397, but not the PARP1 inhibitor olaparib, induced Type I IFN signaling demonstrated by STAT1 phosphorylation. The effect on pSTAT1 was phenocopied by PARP7 knockout (KO). To further demonstrate specificity, we show that simultaneous KO of PARP7 prevented any additional increase of STAT1 phosphorylation by RBN-2397; however, KO of PARP1 had no effect on the induction of Type I IFN signaling by RBN-2397. We had previously reported that RBN-2397 dosing of CT26 tumor bearing immune competent BALB/c mice led to complete and durable tumor regressions which could be reversed by interfering with tumor-derived IFN signaling (1). Here we show that in contrast, RBN-2397 showed modest activity with no tumor regressions in CT26-tumor bearing immunodeficient NOG mice. To assess which immune cell populations are involved in the antitumor effects of RBN-2397, CT26 tumor-bearing BALB/c mice were depleted of CD4 T, CD8 T or NK cells. Depletion of CD4 T or NK cells had no effect on RBN-2397 antitumor activity; however, depletion of CD8 T cells significantly reversed the effects of RBN-2397, suggesting that CD8 T cells are responsible for much of the antitumor immunity induced by RBN-2397. We have discovered and developed RBN-2397, a first-in-class, potent and selective inhibitor of PARP7. We show RBN-2397 restores Type I IFN signaling in cancer cells and that this is an on-target effect of inhibiting the catalytic activity of PARP7 and not PARP1. We further show that the adaptive immune response was required for the antitumor effects of RBN-2397. RBN-2397 is the first agent to enter clinical trials that targets this tumor-intrinsic vulnerability, and a Phase I clinical trial is underway (NCT04053673). (1) AACR Jun 22-24, 2020: Cancer Res 2020;80 (16 Suppl): Abstract nr 3405. Citation Format: Joseph M. Gozgit, Melissa M. Vasbinder, Ryan P. Abo, Kaiko Kunii, Kristy G. Kuplast-Barr, Bin Gui, Alvin Z. Lu, Jennifer R. Molina, Elena Minissale, Kerren K. Swinger, Tim J. Wigle, Danielle J. Blackwell, Christina R. Majer, Yue Ren, Mario Niepel, Ellen Bamberg, Jan-Rung Mo, David Church, Jeff Song, Luke Utley, Patricia E. Rao, Timothy J. Mitchison, Kevin W. Kuntz, Victoria M. Richon, Heike Keilhack. RBN-2397: A potent and selective small molecule inhibitor of PARP7 that induces tumor-derived antitumor immunity dependent on CD8 T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 48.

PARP14 is an interferon-stimulated gene that is overexpressed in multiple tumor types and has been shown to promote the pro-tumor M2 polarization of macrophages and support Th2/Th17 signaling in models of allergic airway disease. PARP14 is a large 203 kDa protein that possesses a catalytic domain responsible for the transfer of mono-ADP-ribose to its substrates, three macrodomains that bind mono-ADP-ribose, a WWE domain that serves as a binding module for poly-ADP-ribose, and an RNA recognition motif. We have previously shown that the potent and reversible enzymatic inhibitor, RBN012759 (IC50 < 0.003 μM, 300-fold selective over monoPARPs, > 1,000-fold selective over polyPARPs), links PARP14 catalytic inhibition with suppression of the antitumor immune response in human primary macrophages and human kidney cancer explants. While this catalytic inhibitor of PARP14 was able to suppress IL-4-driven pro-tumor gene expression in macrophages, it is unknown what roles the non-enzymatic biomolecular recognition motifs play in the biological function of PARP14. To further understand this, we describe a heterobifunctional small molecule, RBN012811, based on a catalytic inhibitor of PARP14 that binds in the enzyme's NAD+-binding site and recruits the E3 ligase cereblon to ubiquitinate PARP14 and selectively target it for degradation. RBN012811 has a IC50 of 0.01 μM against PARP14 in a biophysical assay and is at least 200-fold selective over all other PARPs. In KYSE-270 cancer cells, RBN012811 has a half-maximal degradation concentration (DC50) of 0.005 μM and it does not cause degradation of other PARP enzymes. In human primary macrophages PARP14 degradation by RBN012811 led to a dose-dependent decrease of IL-10 release induced by IL-4 stimulation. Our data demonstrates that RBN012811 is a useful tool to enable further exploration of the role of PARP14 in inflammation and cancer. Citation Format: Tim Wigle, Yue Ren, Jennifer Molina, Danielle Blackwell, Laurie Schenkel, Kerren Swinger, Anne Cheug, Ryan Abo, Elena Minissale, Alvin Lu, Christina Majer, William Church, Bryan Dorsey, Mario Niepel, Nicholas Perl, Kristy Kuplast-Barr, Kristen McEachern, Melissa Vasbinder, Heike Keilhack, Kevin Kuntz. Targeted degradation of PARP14 Using a heterobifunctional small molecule [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1348.

PARPs (poly-ADP-ribose polymerases) are a family of enzymes that regulate a wide variety of important cellular processes including cellular stress signaling pathways implicated in inflammation and cancer. PARP14 is a member of the monoPARP sub-family of PARPs, which catalyze transfer of a single ADP ribose unit (mono-ADP-ribosylation or MARylation) to their substrates, unlike their polyPARP counterparts which construct chains of ADP ribose units (poly-ADP-ribosylation or PARylation). PARP14 is an interferon-stimulated gene (ISG) that is overexpressed in tumors compared to normal tissues and has been implicated by genetic knockout studies to promote pro-tumor macrophage polarization and suppress antitumor inflammatory response due to its role in modulating IL-4 and IFN-γ signaling pathways. Efforts to further explore and validate the role of PARP14 catalytic activity in these pathways have been hampered by a lack of potent, highly selective PARP14 inhibitors. Here we describe the discovery of the first such chemical probe, RBN012759, which inhibits PARP14 with an IC50 of 0.003 µM and exhibits >300-fold selectivity over all mono- and polyPARP family members. Medicinal chemistry efforts that began with an unselective micromolar screening hit were enabled by a detailed understanding of the PARP14 and broader PARP family binding pockets. X-ray co-crystal structures of RBN012759 and key early analogs bound to PARP14 clearly illustrate the origins of their potency and selectivity. RBN012759 is a cell permeable, soluble probe that demonstrates robust, dose-dependent stabilization of endogenous PARP14 and inhibition of MARylation in primary human macrophages. RBN012759 reverses IL-4 driven (pro-tumor) gene expression in macrophages, confirming published data generated with PARP14 KO and supporting an immune suppressive role of PARP14 in tumors. Moreover, we demonstrate that PARP14 inhibition in primary human tumor explants can induce an inflammatory mRNA signature similar to immune checkpoint therapy. Summary: Structure-based design of the first potent and highly selective inhibitor of PARP14, RBN012759, is described. The discovery of this chemical probe enabled exploration of the role of PARP14 in macrophage polarization and inflammatory pathways. RBN012759 reverses IL-4 driven gene expression in macrophages and induces an inflammatory mRNA signature in human tumor explants, data which support an immune suppressive role of PARP14 in tumors. Citation Format: Laurie Schenkel, Jennifer Molina, Kerren Swinger, Ryan Abo, Danielle Blackwell, Anne Cheung, William Church, Kristy Kuplast-Barr, Alvin Lu, Elena Minissale, Mario Niepel, Melissa Vasbinder, Tim Wigle, Victoria Richon, Heike Keilhack, Kevin Kuntz. A potent and selective PARP14 inhibitor decreases pro-tumor macrophage function and elicits inflammatory responses in tumor explants [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1038.

Metabolic reprograming is an emerging hallmark of tumor biology and an actively pursued opportunity in discovery of oncology drugs. Extensive efforts have focused on therapeutic targeting of glycolysis, whereas drugging mitochondrial oxidative phosphorylation (OXPHOS) has remained largely unexplored, partly owing to an incomplete understanding of tumor contexts in which OXPHOS is essential. Here, we report the discovery of IACS-010759, a clinical-grade small-molecule inhibitor of complex I of the mitochondrial electron transport chain. Treatment with IACS-010759 robustly inhibited proliferation and induced apoptosis in models of brain cancer and acute myeloid leukemia (AML) reliant on OXPHOS, likely owing to a combination of energy depletion and reduced aspartate production that leads to impaired nucleotide biosynthesis. In models of brain cancer and AML, tumor growth was potently inhibited in vivo following IACS-010759 treatment at well-tolerated doses. IACS-010759 is currently being evaluated in phase 1 clinical trials in relapsed/refractory AML and solid tumors.

Purpose: Although breast cancers are known to be molecularly heterogeneous, their metabolic heterogeneity is less well understood. This study aimed to identify and evaluate metabolic biomarkers in breast cancers and determine their ability to predict outcomes. Methods: mRNA microarray data from breast cancer cell lines were used to identify bimodal genes, those with the highest potential for robust high/low classification in a clinical setting. Using a panel of breast cancer cell lines, expression and activity of the highest scoring bimodal metabolism gene, lactate dehydrogenase B (LDHB), was quantified and associated with glycolytic phenotype. The contribution of LDHB to glycolysis was evaluated using MDA-MB-231 and HCC1937 cell lines with stable lentiviral knockdown of LDHB. mRNA expression of LDHB was evaluated for association with neoadjuvant chemotherapy response within clinical and PAM50-derived subtypes. Results: LDHB was highly expressed in cell lines with glycolytic, basal-like phenotypes. Knockdown of LDHB in cell lines reduced glycolytic dependence, linking LDHB expression directly to metabolic function. Using four independent patient datasets, LDHB mRNA expression was positively associated with basal subtype and negatively associated with luminal and HER2 subtypes. Furthermore, LDHB predicted basal phenotype independently of hormone-receptor (HR) clinical status (OR = 21.6 for HR-positive/HER2-negative and OR = 18.2 for triple-negative). While LDHB expression could predict basal phenotype, high LDHB expression identified aggressive breast cancer tumors that were primarily but not exclusively basal. Importantly, high LDHB expression predicted pathological complete response to neoadjuvant chemotherapy for both hormone receptor (HR) positive/HER2-negative (OR = 4.0, P = .0002) and triple-negative (OR = 3.0, P = .003) cancers. Consistent with increased response to chemotherapy, LDHB expression in basal cancers within the triple-negative group was associated with the proliferative marker CCNB1 (P < .0001). Conclusion: mRNA expression of LDHB as a single marker predicted glycolytic phenotype in cell lines and response to neoadjuvant chemotherapy in breast cancers independently of HR status. These observations support prospective clinical evaluation of LDHB as a predictive marker of response for breast cancer patients treated with neoadjuvant chemotherapy. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-06-06.

The phosphatidylinositol-3-OH kinase (PI3K)-Akt pathway is activated in cancer by genetic or epigenetic events and efforts are under way to develop targeted therapies. phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is the major brake of the pathway and a common target for inactivation in glioblastoma, one of the most aggressive and therapy-resistant cancers. To achieve potent inhibition of the PI3K-Akt pathway in glioblastoma, we need to understand its mechanism of activation by investigating the interplay between its regulators. We show here that PTEN modulates the PI3K-Akt pathway in glioblastoma within a tumor suppressor network that includes Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) and pleckstrin-homology domain leucine-rich repeat protein phosphatases 1 (PHLPP1). The NHERF1 adaptor, previously characterized by our group as a PTEN ligand and regulator, shows also PTEN-independent Akt-modulating effects that led us to identify the PHLPP1/PHLPP2 Akt phosphatases as NHERF1 ligands. NHERF1 interacts via its PDZ domains with PHLPP1/PHLPP2 and scaffolds heterotrimeric complexes with PTEN. Functionally, PHLPP1 requires NHERF1 for membrane localization and growth-suppressive effects. PHLPP1 loss boosts Akt phosphorylation only in PTEN-negative cells and cooperates with PTEN loss for tumor growth. In a panel of low-grade and high-grade glioma patient samples, we show for the first time a significant disruption of all three members of the PTEN-NHERF1-PHLPP1 tumor suppressor network in high-grade tumors, correlating with Akt activation and patient's abysmal survival. We thus propose a PTEN-NHERF1-PHLPP PI3K-Akt pathway inhibitory network that relies on molecular interactions and can undergo parallel synergistic hits in glioblastoma.

Glioblastoma is a deadly brain tumor with a median survival of 1.3 years after surgery, radiotherapy and chemotherapy. The dismal prognosis is due to the propensity of the tumor to invade adjacent normal brain. Although next generation sequencing (NGS) led to genomic landscaping of glioblastoma, there is little insight into the mechanism of neoplastic cell invasion, the detection of the invasive cells or their therapeutic targeting. To address these critical issues, we developed a murine model of invasive glioblastoma that mimics the human disease. Using a cancer stem-cell like human glioblastoma cell line in an orthotopic model, we isolated pairs of neoplastic cells growing either in the core of the tumor, or away for the core, in invasive secondary foci. A phenotypic assessment showed significantly decreased animal survival, increased cancer stemness and invasion in the invasive cells in comparison to core cells. Expression profiling of Core and Inv cells resulted in the identification of differentially expressed gene targets controlling metabolic pathways, cell-cell and cell-matrix adhesion and cell differentiation. A 300 gene library, including these targets, was designed for NGS of high grade diffuse glioma cases. In a series of 4 pediatric and adult WHO grade IV diffuse glioma autopsy cases with a total of 28 sequenced distinct primary and secondary tumor foci, as well as normal brain, we found mutations in several of the genes identified in the mouse model. Two categories of novel targets potentially relevant for therapy were confirmed and further addressed: extracellular matrix proteases and metabolic enzymes. These translational findings and the implications for the molecular diagnosis and therapy of glioblastoma will be discussed.

Over the past few years we and others reported that specific populations of tumor cells including AML, subsets of lymphoma, glioblastoma, triple negative breast cancer (TNBC), melanoma and pancreatic ductal adenocarcinoma (PDAC) are highly dependent upon oxidative phosphorylation (OXPHOS) to meet their energy and biomass needs. Inhibition of OXPHOS in the context of these dependent tumor populations represents therefore an exciting therapeutic opportunity. Through an extensive medicinal chemistry campaign we discovered IACS-010759, a potent, selective small molecule inhibitor of complex I of the mitochondria electron transport chain that possesses excellent pharmacokinetic (PK) and pharmacologic properties, making it suitable for clinical development. We advanced IACS-010759 through IND studies and have recently initiated Phase I studies in patients with relapsed/refractory acute myeloid leukemia (AML) and advanced solid tumors and lymphomas (NCT02882321 and NCT03291938). In this presentation we will describe the identification of a novel series of Complex I inhibitors and their optimization into the clinical candidate compound IACS-010759. Several challenges were successfully overcome, including the optimization of the pharmacokinetic profile and the identification of inhibitors with minimal activity shift across preclinical species, thus enabling a thorough evaluation of the efficacy and toxicology profile. Aspects of the extensive translational research conducted to elucidate the mechanism of action of IACS-010759 and to position it into the clinic will be discussed, including the compelling pharmacological response observed in multiple PDX models of primary AML, and PDX xenograft models of lymphoma, TNBC, glioblastoma, melanoma and PDAC. The observed response was associated with robust pharmacodynamic read-out as assessed by modulation of oxygen consumption rate (OCR), aspartate and specific transcriptional changes. The presentation will also cover the preclinical development activities which resulted in IACS-010759 advancing into on-going phase 1 evaluation in AML and solid tumors. Citation Format: Maria Emilia Di Francesco, Joseph R. Marszalek, Timothy McAfoos, Christopher L. Carroll, Zhijun Kang, Gang Liu, Jay P. Theroff, Jennifer P. Bardenhager, Madhavi L. Bandi, Jennifer R. Molina, Sonal Gera, Marina Protopopova, Yuting Sun, Mary K. Geck Do, Ningping Feng, Jason P. Gay, Florian Muller, Marina Konopleva, Funda Meric-Bernstam, Carlo Toniatti, Timothy P. Heffernan, Giulio F. Draetta, Philip Jones. Discovery and development of IACS-010759, a novel inhibitor of Complex I currently in phase I studies to exploit oxidative phosphorylation dependency in acute myeloid leukemia and solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1655.

Glioblastoma is the most frequent and malignant brain tumor, characterized by an elevated capacity for cellular proliferation and invasion. Recently, it was demonstrated that podoplanin membrane sialo-glycoprotein encoded by PDPN gene is over-expressed and related to cellular invasion in astrocytic tumors; however the mechanisms of regulation are still unknown. MicroRNAs are noncoding RNAs that regulate gene expression and several biological processes and diseases, including cancer. Nevertheless, their roles in invasion, proliferation, and apoptosis of glioblastoma are not completely understood. In this study, we focused on miR-29b and miR-125a, which were predicted to regulate PDPN, and demonstrated that these microRNAs directly target the 3′ untranslated region of PDPN and inhibit invasion, apoptosis, and proliferation of glioblastomas. Furthermore, we report that miR-29b and miR-125a are downregulated in glioblastomas and also in CD133-positive cells. Taken together, these results suggest that miR-29b and miR-125a represent potential therapeutic targets in glioblastoma. © 2010 Wiley-Liss, Inc.

Cell culture is an essential tool in drug discovery, tissue engineering and stem cell research. Conventional tissue culture produces two-dimensional cell growth with gene expression, signalling and morphology that can be different from those found in vivo, and this compromises its clinical relevance1,2,3,4,5. Here, we report a three-dimensional tissue culture based on magnetic levitation of cells in the presence of a hydrogel consisting of gold, magnetic iron oxide nanoparticles and filamentous bacteriophage. By spatially controlling the magnetic field, the geometry of the cell mass can be manipulated, and multicellular clustering of different cell types in co-culture can be achieved. Magnetically levitated human glioblastoma cells showed similar protein expression profiles to those observed in human tumour xenografts. Taken together, these results indicate that levitated three-dimensional culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and may be more feasible for long-term multicellular studies. Magnetic levitation of cells with a hydrogel containing magnetic nanoparticles forms a three-dimensional tissue culture suited for various multicellular studies.

Glioblastoma is a frequent brain malignancy with a dismal prognosis. The molecular changes causing its aggressive phenotype are under investigation. We report that the cytoskeletal-related proteins neurofibromatosis type 2 (NF2) and ezrin have opposite yet interdependent activities in glioblastoma growth. We show that NF2 is absent in approximately one-third of glioblastoma cell lines and tumors, and that it suppresses growth when expressed in cells. Although ezrin overexpression was previously observed in glioblastoma, we show here that ezrin enhanced cell proliferation and anchorage-independent growth but only in cells expressing NF2. Ezrin interacted and delocalized NF2 from the cortical compartment releasing its inhibition on Rac1. By using swap NF2-ezrin molecules, we identified that the opposite effects on cell growth of NF2 and ezrin depend on their amino-terminal FERM domain. The subcellular cortical localization appeared important for NF2 suppressive activity. In contrast, the ability of ezrin to enhance growth or complex NF2 did not depend on the molecular conformation or subcellular localization. In conclusion, these studies show 2 mechanisms for NF2 inactivation in glioblastoma: (i) decreased protein expression and (ii) increasing dosages of ezrin that disable NF2 by intermolecular association and aberrant intracellular recruitment.

Objective— Susceptibility to atherosclerosis is genetically complex, and modifier genes that do not operate via traditional risk factors are largely unknown. A mouse genetics approach can simplify the genetic analysis and provide tools for mechanistic studies. Methods and Results— We previously identified atherosclerosis susceptibility QTL ( Athsq1 ) on chromosome 4 acting independently of systemic risk factors. We now report confirmation of this locus in congenic strains carrying the MOLF-derived susceptibility allele in the C57BL/6J- Ldlr −/− genetic background. Homozygous congenic mice exhibited up to 4.5-fold greater lesion area compared to noncongenic littermates ( P Athsq1 congenic interval contains the mouse region homologous to a widely-replicated CHD locus on human chromosome 9p21. Conclusion— These studies confirm the proatherogenic activity of a novel gene(s) in the MOLF-derived Athsq1 locus and provide in vivo evidence for a causative role of versican in lesion development.

Rab25, by altering trafficking of critical cellular resources, influences cell metabolism and survival during stress conditions. Overall, perturbations in the vesicular trafficking machinery change cellular bioenergetics that can be directly measured in real time as Oxygen Consumption Rate, OCR (mitochondrial respiration) and Extracellular Acidification Rate, ECAR (glycolysis) by an extracellular flux analyzer (XF96, Seahorse Biosciences, MA). Additionally, overall turnover of glucose, lactate, as well as glutamine and glutamate can be measured biochemically using the YSI2900 Biochemistry Analyzer (YSI Incorporated, Life Sciences, OH). A combination of these two methods allows a precise and quantitative approach to interrogate the role of Rab25 as well as other Rab GTPases in central carbon energy metabolism.

Purpose: Although breast cancers are known to be molecularly heterogeneous, their metabolic phenotype is less well-understood and may predict response to chemotherapy. This study aimed to evaluate metabolic genes as individual predictive biomarkers in breast cancer. Experimental Design: mRNA microarray data from breast cancer cell lines were used to identify bimodal genes—those with highest potential for robust high/low classification in clinical assays. Metabolic function was evaluated in vitro for the highest scoring metabolic gene, lactate dehydrogenase B (LDHB). Its expression was associated with neoadjuvant chemotherapy response and relapse within clinical and PAM50-derived subtypes. Results: LDHB was highly expressed in cell lines with glycolytic, basal-like phenotypes. Stable knockdown of LDHB in cell lines reduced glycolytic dependence, linking LDHB expression directly to metabolic function. Using patient datasets, LDHB was highly expressed in basal-like cancers and could predict basal-like subtype within clinical groups [OR = 21 for hormone receptor (HR)-positive/HER2-negative; OR = 10 for triple-negative]. Furthermore, high LDHB predicted pathologic complete response (pCR) to neoadjuvant chemotherapy for both HR-positive/HER2-negative (OR = 4.1, P < 0.001) and triple-negative (OR = 3.0, P = 0.003) cancers. For triple-negative tumors without pCR, high LDHB posttreatment also identified proliferative tumors with increased risk of recurrence (HR = 2.2, P = 0.006). Conclusions: Expression of LDHB predicted response to neoadjuvant chemotherapy within clinical subtypes independently of standard prognostic markers and PAM50 subtyping. These observations support prospective clinical evaluation of LDHB as a predictive marker of response for patients with breast cancer receiving neoadjuvant chemotherapy. Clin Cancer Res; 19(13); 3703–13. ©2013 AACR.

Tumor cells depend on both glycolysis and oxidative phosphorylation (OXPHOS) for energy and biomass production leading to robust cell proliferation. Recent data has demonstrated a dependence of various tumor types on mitochondrial OXPHOS, which represents an exciting therapeutic opportunity. Through an extensive medicinal chemistry campaign, IACS-10759 was identified as a potent, selective inhibitor of complex I of the electron transport chain, which is orally bioavailable and has excellent PK and physicochemical properties in preclinical species. Our group and others have demonstrated that a variety of tumor types including: AML, plus subsets of lymphoma, breast, melanoma and PDAC are highly dependent on OXPHOS to meet energy and biomass demands. Treatment of multiple cell lines and patient derived xenograft (PDX) models in multiple cancer types with IACS-10759 led to decreased oxygen consumption rate (OCR). IACS-10759 treatment also led to a robust decrease in cell viability and often an increase in apoptosis with EC50 values between 1 nM - 50 nM across multiple lines. Through a series of mechanistic studies we established that IACS-10759 blocks complex I of the electron transport at the quinone binding site. In an orthotopic xenograft model of primary AML cells derived from a patient who was refractory to standard of care and salvage therapies, 42 days of IACS-10759 treatment with 3 and 10 mg/kg orally using a 5 on/2 off schedule extended the median survival by greater than 2-fold. Efficacy was paralleled by robust modulation of OCR, aspartate, and p-AMPK levels. Additionally, tumor growth inhibition or regression was also observed in cell line and PDX xenograft models of lymphoma, triple negative breast, melanoma and PDAC treated with IACS-10759, indicating that subsets of several non-AML indications are also dependent on OXPHOS. Mechanistically, extensive metabolic profiling and flux analysis revealed that the response to IACS-10759 was associated with induction of a metabolic imbalance that negatively impacted energy homeostasis, amino acid biosynthesis, and NTP production due to reduced conversion of NADH to NAD+ by complex I, decreased ATP production, TCA cycle flux and nucleotide biosynthesis. As a result of the robust response in multiple cell lines, primary patient samples, and efficacy in PDX models, IACS-10759 has been advanced through IND enabling studies. GLP safety and toxicology have been completed, and we expect to file an IND at the end of 1Q2016 and initiate a Phase I clinical trial in AML during the second quarter of 2016. Citation Format: Jennifer R. Molina, Marina Protopopova, Madhavi Bandi, Jennifer Bardenhagen, Christopher Bristow, Christopher Carroll, Edward Chang, Ningping Feng, Jason Gay, Mary Geck Do, Jennifer Greer, Sha Huang, Yongying Jiang, Marina Konopleva, Polina Matre, Jing Han, Zhijun Kang, Gang Liu, Timothy McAfoos, Pietro Morlacchi, Melinda Smith, Sonal Gera, Jay Theroff, Quanyun Xu, Juliana Velez, Carlo Toniatti, Timothy Heffernan, Giulio Draetta, M. Emilia Di Francesco, Philip Jones, Joseph R. Marszalek. Title: IACS-010759 is a novel clinical candidate that targets AML cells by inducing a metabolic catastrophe through inhibition of oxidative phosphorylation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 335.

Apoptosis culminates in secondary necrosis due to lack of ATP. Cancer stem cells form spheres after apoptosis by evoking the blebbishield emergency program. Hence, determining how blebbishields avoid secondary necrosis is crucial. Here we demonstrate that N-Myc and VEGFR2 control transformation from blebbishields, during which oligomers of K-Ras, p27, BAD, Bax, and Bak boost glycolysis to avoid secondary necrosis. Non-apoptotic cancer cells also utilize oligomers to boost glycolysis, which differentiates the glycolytic function of oligomers from their apoptotic action. Smac mimetic in combination with TNF-α or TRAIL but not in combination with FasL abrogates transformation from blebbishields by inducing secondary necrosis. Thus blebbishield-mediated transformation is dependent on glycolysis, and Smac mimetics represent potential candidates to abrogate the blebbishield emergency program.

Motivation: Protein signaling networks play a key role in cellular function, and their dysregulation is central to many diseases, including cancer. To shed light on signaling network topology in specific contexts, such as cancer, requires interrogation of multiple proteins through time and statistical approaches to make inferences regarding network structure. Results: In this study, we use dynamic Bayesian networks to make inferences regarding network structure and thereby generate testable hypotheses. We incorporate existing biology using informative network priors, weighted objectively by an empirical Bayes approach, and exploit a connection between variable selection and network inference to enable exact calculation of posterior probabilities of interest. The approach is computationally efficient and essentially free of user-set tuning parameters. Results on data where the true, underlying network is known place the approach favorably relative to existing approaches. We apply these methods to reverse-phase protein array time-course data from a breast cancer cell line (MDA-MB-468) to predict signaling links that we independently validate using targeted inhibition. The methods proposed offer a general approach by which to elucidate molecular networks specific to biological context, including, but not limited to, human cancers. Availability: http://mukherjeelab.nki.nl/DBN (code and data). Contact: s.hill@nki.nl; gmills@mdanderson.org; s.mukherjee@nki.nl Supplementary information: Supplementary data are available at Bioinformatics online.

Interactions of leukemia cells and their bone marrow (BM) microenvironment are known to play a key role in the survival and growth of leukemic cells. It has been postulated that specific niches provide a sanctuary where subpopulations of leukemic cells evade chemotherapy-induced death and acquire a drug-resistant phenotype. Understanding the cellular and molecular biology of the leukemia stem cell (LSC) niche and of microenvironment/leukemia interactions may provide new targets that allow destruction of LSCs without adversely affecting normal stem cell self-renewal. Key emerging therapeutic targets include chemokine receptors such as CXCR4 and hypoxia-related proteins, as well as the metabolic abnormalities of the leukemia-associated stroma. We have recently reported that CXCR4 inhibition causes leukemia cell dislodgement from CXCL12-producing marrow niches, reduced proliferation and induction of differentiation of AML cells in an in vivo model of AML, translating into pronounced anti-leukemia effects. Studies in murine leukemia models using the hypoxia probe pimonidazole demonstrated extensive areas of hypoxia within leukemic, but not healthy normal, bone marrow. Time-course analysis of bone marrow spaces within calvaria and femurs by multiphoton intravital microscopy (MP-IVM) demonstrated lodging of p190-Bcr/Abl tdTomato cells in close proximity to blood vessels, followed by accumulation of leukemia cells localized within the sinusoidal and marrow spaces resulting in the demise of the animals within 3 weeks. In this model, pimonidazole detected hypoxic areas despite abundant vascular supply in the marrow cavities. In vivo magnetic resonance imaging with hyperpolarized pyruvate showed higher pyruvate-lactate conversion (high glycolytic flux) in leukemic marrows. These findings were supported by significant pimonidazole uptake by the diseased bone marrow in patients with acute leukemia, causing stabilization of HIF-1α in 55% (76/138) of primary AML patients and of its target CA9. Paradoxically, AML cells become highly dependent on mitochondrial oxidative phosphorylation (OXPHOS) for their survival, and inhibition of OXPHOS with the novel small molecule complex I inhibitor IACS-10759 inhibits oxygen consumption, eliminates hypoxia in vivo and inhibits AML growth. These findings suggest that altered tumor metabolism underlies the hypoxia observed in leukemias. We postulate that the altered tumor microenvironment within the hypoxic niche cells will influence leukemia development and response to therapy. Hence, targeting key metabolic alterations should be considered in the armamentarium of anti-AML therapies. IACS-10759 is presently completing IND enabling safety/toxicity studies with first in human studies targeting relapsed/refractory AML planned for 2016. Citation Format: Marina Y. Konopleva, Tomasz Zal, Niki M. Zacharias Millward, Byoung-Sik Cho, Karine Harutyunyan, Anna Zal, Hong Mu, Sergej Konoplev, Juliana Benito, Juliana Velez, Carlos Bueso-Ramso, Jennifer Molina, Pratip K. Bhattacharya, Maria Emilia Di Francesco, Joseph Marszalek, Michael Andreeff. Altered metabolism in leukemic microenvironment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PL07-01.

Acute myeloid leukemia (AML) is a highly aggressive disease with a high mortality rate that encompasses several genetically and clinically diverse hematological malignancies characterized by clonal expansion of transformed stem/progenitor cells with limited ability to differentiate into mature blood cells. Standard of care for AML has progressed minimally in the past 30 years for relapse/refractory AML, with survival rates of 65 years. Therefore, novel, highly effective therapeutics are needed for this population. Targeting bioenergetic susceptibilities is an exciting area of oncology therapeutics that is potentially applicable in AML. Our group and others have shown that AML blasts depend significantly on mitochondrial oxidative phosphorylation to meet their energy and biomass production demands. Through an extensive medicinal chemistry campaign IACS-10759 was identified as a potent, selective inhibitor of complex I of the electron transport chain with excellent PK and a suitable overall profile. In AML cell lines and primary AML blasts treated ex vivo, we observe a robust decrease in proliferation and a concomitant increase in apoptosis with EC50 values of less than 10 nM. Response to IACS-10759 in AML cells was associated with induction of a metabolic catastrophe that negatively impacted the cells' ability to sustain energy homeostasis, amino acid biosynthesis, and nucleotide production. In a primary AML patient derived xenograft model from a patient who was refractory to standard of care and salvage therapies, 42 days of IACS-10759 (QDx5/week) treatment at 10 mg/kg extended the median survival by greater than 2-fold. Inhibition of OXPHOS by IACS-10759 was confirmed in AML cell lines and PDX models by a decrease in oxygen consumption and significant changes in gene and protein expression, non-essential amino acids and nucleotides. Due to the robust response in AML cell lines, primary AML samples ex vivo, and in vivo efficacy in primary AML PDX models, IACS-10759 has been advanced through IND enabling studies with first-in-human studies targeted for the second quarter of 2016. Citation Format: Jennifer R. Molina, Marina Protopopova, Madhavi Bandi, Jennifer Bardenhagen, Christopher Bristow, Maria Alimova, Christopher Carroll, Edward Chang, Ningping Feng, Jason Gay, Mary Geck Do, Jennifer Greer, Sha Huang, Yongying Jiang, Marina Konopleva, Polina Matre, Zhijun Kang, Gang Liu, Timothy McAfoos, Pietro Morlacchi, Melinda Smith, Sonal Sonal, Jay Theroff, Quanyun Xu, Giulio Draetta, Philip Jones, Carlo Toniatti, M. Emilia Di Francesco, Joseph R. Marszalek. IACS-010759 is a novel inhibitor of oxidative phosphorylation that selectively targets AML cells by inducing a metabolic catastrophe. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A15.

Glioblastoma multiforme (GBM) is a severe brain malignancy with limited treatment and dismal prognosis. The tumor suppressor PTEN, a major inhibitor of the phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway, is frequently deleted in GBM tumors. PTEN antagonizes PI3K by dephosphorylating PI3K phosphoinositide substrates at the plasma membrane. The PTEN binding adapter protein NHERF1/EBP50 is overexpressed in GBM but its effects on tumorigenesis have yet to be determined. Here, we show that NHERF1 is localized to the plasma membrane in normal astrocytes and to the cytoplasm of GBM tumor cells. This cytoplasmic shift paralleled an altered membrane distribution of wild-type PTEN with consecutive Akt activation. Membrane re-targeting of NHERF1 in GBM cells recruited PTEN to the membrane and suppressed Akt activation and cell proliferation. Conversely, NHERF1 depletion in GBM cells with membrane-localized NHERF1 increased cell proliferation and Akt activation. Our findings define a tumor suppressor role for NHERF1 at the plasma membrane, and reveal a novel mechanism for PI3K/Akt activation through PTEN inactivation caused by a loss of membrane-localized NHERF1. Cancer Res; 70(17); 6697–703. ©2010 AACR.

Glioblastoma multiforme (GBM) is the most frequent and most aggressive brain tumor in adults. The dismal prognosis is due to postsurgery recurrences arising from escaped invasive tumor cells. The signaling pathways activated in invasive cells are under investigation, and models are currently designed in search for therapeutic targets. We developed here an in vivo model of human invasive GBM in mouse brain from a GBM cell line with moderate tumorigenicity that allowed simultaneous primary tumor growth and dispersal of tumor cells in the brain parenchyma. This strategy allowed for the first time the isolation and characterization of matched sets of tumor mass (Core) and invasive (Inv) cells. Both cell populations, but more markedly Inv cells, acquired stem cell markers, neurosphere renewal ability, and resistance to rapamycin-induced apoptosis relative to parental cells. The comparative phenotypic analysis between Inv and Core cells showed significantly increased tumorigenicity in vivo and increased invasion with decreased proliferation in vitro for Inv cells. Examination of a large array of signaling pathways revealed extracellular signal-regulated kinase (Erk) down-modulation and Akt activation in Inv cells and an opposite profile in Core cells. Akt activation correlated with the increased tumorigenicity, stemness, and invasiveness, whereas Erk activation correlated with the proliferation of the cells. These results underscore complementary roles of the Erk and Akt pathways for GBM proliferation and dispersal and raise important implications for a concurrent inhibitory therapy.

This review summarizes the emerging roles of NHERF1/EBP50 adaptor protein in tumorigenesis. NHERF1/EBP50 (Na(+)/H(+) exchanger regulating factor 1; ezrin-radixin-moesin (ERM) binding phosphoprotein of 50 kDa) is a PDZ domain-containing protein with physiological localization at the plasma membrane. We discuss in this review the functions of NHERF1/EBP50 as a linker between membrane proteins and the cytoskeleton network, as well as its involvement in different types of cancer, such as breast and liver cancers. Recent evidence obtained from our laboratory and from other groups shows that NHERF1/EBP50 is an important player in cancer progression. It appears that, depending on its subcellular distribution, NHERF1/EBP50 may behave either as a tumor suppressor, when it is localized at the plasma membrane, or as an oncogenic protein, when it is shifted to the cytoplasm. We provide here an overview of the mechanisms by which this adaptor protein controls cell transformation, and propose a model suggesting a dual role of NHERF1/EBP50 in cancer.

ABCA1, the mutant molecule in Tangier Disease, mediates efflux of cellular cholesterol to apoA-I and is induced by liver X receptor (LXR)/retinoid X receptor (RXR) transcription factors. Retinoic acid receptor (RAR) activators (all-trans-retinoic acid [ATRA] and TTNPB) were found to increase ATP-binding cassette transporter 1 (ABCA1) mRNA and protein in macrophages. In cellular cotransfection assays, RARgamma/RXR activated the human ABCA1 promoter, via the same direct repeat 4 (DR4) promoter element as LXR/RXR. Chromatin immunoprecipitation analysis in macrophages confirmed the binding of RARgamma/RXR to the ABCA1 promoter DR4 element in the presence of ATRA, with weaker binding of RARalpha/RXR, and no binding of RARbeta/RXR. However, in macrophages from RARgamma(-/-) mice, TTNPB still induced ABCA1, in association with marked upregulation of RARalpha, suggesting that high levels of RARalpha can compensate for the absence of RARgamma. Dose-response experiments with ATRA in mouse primary macrophages showed that other LXR target genes were weakly induced (ABCG1 and SREBP-1c) or not induced (apoE and LXRalpha). The more specific RAR activator TTNPB did not induce SREBP-1c in mouse primary macrophages or liver. These studies indicate a direct role of RARgamma/RXR in induction of macrophage ABCA1.

Background: Several distinct subtypes of breast cancer (Luminal, Basal-like and Her-2+) have been identified by gene expression profiling of breast cancers and cell lines. Although much is known about the regulation of cell signaling in each breast cancer subtype, little is known about subtype-specific energy metabolism and its regulation. The majority of cancers, including breast cancer, acquire an accelerated metabolic index as part of the transformation process. One of the most studied metabolic changes in cancer, referred to as the Warburg effect, is the increased uptake of glucose and its conversion to lactate; which is released from the cell, thus creating an astringent tumor microenvironment with high lactate and low pH. Accumulation of lactate in the tumor microenvironment presents cancer cells with a potential rich carbon source that could be exploited when the preferred nutrient sources, glucose and glutamine, are not abundant or available. Thus uptake and conversion of lactate to pyruvate and then entry into the TCA cycle and oxidative phosphorylation could generate energy that could potentially allow cancer cells to survive until other nutrients become available or until the cancer cells can invade and migrate toward nutrient rich environments, in other words, until they spread locally and metastasize. Methods and Results: Examination of 59 breast cancer cell lines shows differences in expression of numerous proteins and enzymes involved in cellular metabolism, including the lactate transporters (MCT), the enzyme lactate dehydrogenase (LDH), and glucose transporter proteins (GLUT). We found that MCT1 expression is lost in the Luminal subtype and correlates with loss of LDHB and the regulator and chaperone of MCT1, CD147 (Basigin). Conversely, MCT1 is highly expressed in Basal-like and normal cell lines, along with CD147 and LDHB. While monocarboxylate transporter proteins can transport bi-directionally, MCT1 preferentially transports lactate into the cell, while MCT4 transports lactate out of the cell. The loss of MCT1 in luminal cells suggests a distinct energy metabolism in this subtype versus basal-like cells. The basal-like subtype has been further categorized into Basal and Claudin-low subtypes. Claudin-low cells express stem-like markers such as CD44+/CD24-, EMT markers such as vimentin, and are low expressers of the Claudin proteins. We found that MCT1 and CD147 are highly expressed and differentially regulated in Claudin-low cell lines as compared to normal cells. We have evidence that high lactate (10mM) as a sole energy source delays ATP loss and apoptosis in MCT1 expressing cells, but not in luminal cell lines that lack MCT1. Thus, claudin-low tumors may benefit from local lactate production providing an unexpected energy source. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-05-03.

Gene expression data of breast cancer cell lines and patient tumors reveal several distinct subtypes that demonstrate different metabolic phenotypes. Our goal was to determine what metabolism genes contribute to the metabolic phenotypes. We hypothesized that dichotomous expression of metabolism genes would identify key drivers of cell metabolism. To select candidate genes, we completed a bimodal analysis using microarray data from 59 breast cancer cell lines. To generate a metabolic network, dichotomous genes were selected from known or probable metabolic networks including electron transport, TCA cycle, nutrient transport, pentose phosphate shunt, energy requiring pathways, and glycolysis. Two groups of dichotomously expressed genes emerged from this analysis: TCA cycle anaplerotic genes (including PFKP, GLS) and cataplerotic/biosynthetic genes (including FASN, GLUL, and FBP1). The gene with the highest bimodal index was LDHB that encodes lactate dehydrogenase B. For breast cancer cell lines, LDHB mRNA and protein were most highly expressed in ER-, glycolytic cell lines. LDHB is reported by others to be part of a “MYC signature,” and consistent with this hypothesis, for ER- cell lines, LDHB mRNA levels were highly associated with MYC and glutaminase, a metabolic down-stream target of MYC. For patient tumors, LDHB mRNA expression was also bimodal with the highest expression in basal-like cancers. Elevated expression of LDHB mRNA predicted worse clinical outcomes for patients with Her2 positive and triple negative tumors. Compared to normal-like breast tissue from patients, LDHB mRNA expression was elevated in triple negative tumors. We conclude that LDHB may be a target of MYC and contribute to the glycolytic phenotype of triple-negative tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1273. doi:10.1158/1538-7445.AM2011-1273

Background Cancer progression occurs upon mutations on regulatory genes that control biological functions including cellular bioenergetics. Such perturbations lead to a metabolic switch that favors aerobic glycolysis and lactate production over oxidative phosphorylation. This process is known as the Warburg effect and results in a lactate-rich tumor microenvironment. The apparently wasteful mechanism has raised the question of why cancer cells switch from the high ATP producing TCA cycle/OXPHOS to glycolysis; and whether lactate serves a biological function in energy metabolism since a high lactate environment correlates with worse patient prognosis in several malignancies including breast cancer. Our goals were to first, determine the fate of carbons from lactate in breast cancer cells; and second, determine the mechanism of lactate metabolism.