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Supplementary Figures S1-S6 and Tables S1-S4

Supplementary Materials and Methods

Targeting carbonic anhydrase IX acidifies intracellular pH, disrupts redox homeostasis, and creates vulnerability to ferroptosis., The metabolic mechanisms involved in the survival of tumor cells within the hypoxic niche remain unclear. We carried out a synthetic lethal CRISPR screen to identify survival mechanisms governed by the tumor hypoxia–induced pH regulator carbonic anhydrase IX (CAIX). We identified a redox homeostasis network containing the iron-sulfur cluster enzyme, NFS1. Depletion of NFS1 or blocking cyst(e)ine availability by inhibiting xCT, while targeting CAIX, enhanced ferroptosis and significantly inhibited tumor growth. Suppression of CAIX activity acidified intracellular pH, increased cellular reactive oxygen species accumulation, and induced susceptibility to alterations in iron homeostasis. Mechanistically, inhibiting bicarbonate production by CAIX or sodium-driven bicarbonate transport, while targeting xCT, decreased adenosine 5′-monophosphate–activated protein kinase activation and increased acetyl–coenzyme A carboxylase 1 activation. Thus, an alkaline intracellular pH plays a critical role in suppressing ferroptosis, a finding that may lead to the development of innovative therapeutic strategies for solid tumors to overcome hypoxia- and acidosis-mediated tumor progression and therapeutic resistance.

Background & Aims Most pancreatic ductal adenocarcinomas (PDACs) express an activated form of KRAS, become hypoxic and dysplastic, and are refractory to chemo and radiation therapies. To survive in the hypoxic environment, PDAC cells upregulate enzymes and transporters involved in pH regulation, including the extracellular facing carbonic anhydrase 9 (CA9). We evaluated the effect of blocking CA9, in combination with administration of gemcitabine, in mouse models of pancreatic cancer. Methods We knocked down expression of KRAS in human (PK-8 and PK-1) PDAC cells with small hairpin RNAs. Human and mouse (KrasG12D/Pdx1-Cre/Tp53/RosaYFP) PDAC cells were incubated with inhibitors of MEK (trametinib) or extracellular signal-regulated kinase (ERK), and some cells were cultured under hypoxic conditions. We measured levels and stability of the hypoxia-inducible factor 1 subunit alpha (HIF1A), endothelial PAS domain 1 protein (EPAS1, also called HIF2A), CA9, solute carrier family 16 member 4 (SLC16A4, also called MCT4), and SLC2A1 (also called GLUT1) by immunoblot analyses. We analyzed intracellular pH (pHi) and extracellular metabolic flux. We knocked down expression of CA9 in PDAC cells, or inhibited CA9 with SLC-0111, incubated them with gemcitabine, and assessed pHi, metabolic flux, and cytotoxicity under normoxic and hypoxic conditions. Cells were also injected into either immune-compromised or immune-competent mice and growth of xenograft tumors was assessed. Tumor fragments derived from patients with PDAC were surgically ligated to the pancreas of mice and the growth of tumors was assessed. We performed tissue microarray analyses of 205 human PDAC samples to measure levels of CA9 and associated expression of genes that regulate hypoxia with outcomes of patients using the Cancer Genome Atlas database. Results Under hypoxic conditions, PDAC cells had increased levels of HIF1A and HIF2A, upregulated expression of CA9, and activated glycolysis. Knockdown of KRAS in PDAC cells, or incubation with trametinib, reduced the posttranscriptional stabilization of HIF1A and HIF2A, upregulation of CA9, pHi, and glycolysis in response to hypoxia. CA9 was expressed by 66% of PDAC samples analyzed; high expression of genes associated with metabolic adaptation to hypoxia, including CA9, correlated with significantly reduced survival times of patients. Knockdown or pharmacologic inhibition of CA9 in PDAC cells significantly reduced pHi in cells under hypoxic conditions, decreased gemcitabine-induced glycolysis, and increased their sensitivity to gemcitabine. PDAC cells with knockdown of CA9 formed smaller xenograft tumors in mice, and injection of gemcitabine inhibited tumor growth and significantly increased survival times of mice. In mice with xenograft tumors grown from human PDAC cells, oral administration of SLC-0111 and injection of gemcitabine increased intratumor acidosis and increased cell death. These tumors, and tumors grown from PDAC patient-derived tumor fragments, grew more slowly than xenograft tumors in mice given control agents, resulting in longer survival times. In KrasG12D/Pdx1-Cre/Tp53/RosaYFP genetically modified mice, oral administration of SLC-0111 and injection of gemcitabine reduced numbers of B cells in tumors. Conclusions In response to hypoxia, PDAC cells that express activated KRAS increase expression of CA9, via stabilization of HIF1A and HIF2A, to regulate pH and glycolysis. Disruption of this pathway slows growth of PDAC xenograft tumors in mice and might be developed for treatment of pancreatic cancer.

The architectural complexity and heterogeneity of the tumor microenvironment (TME) remains a substantial obstacle in the successful treatment of cancer. Hypoxia, caused by insufficient oxygen supply, and acidosis, resulting from the expulsion of acidic metabolites, are prominent features of the TME. To mitigate the consequences of the hostile TME, cancer cells metabolically rewire themselves and express a series of specific transporters and enzymes instrumental to this adaptation. One of these proteins is carbonic anhydrase (CA)IX, a zinc-containing extracellular membrane bound enzyme that has been shown to play a critical role in the maintenance of a neutral intracellular pH (pHi), allowing tumor cells to survive and thrive in these harsh conditions. Although CAIX has been considered a promising cancer target, only two antibody-based therapeutics have been clinically tested so far. To fill this gap, we generated a series of novel monoclonal antibodies (mAbs) that specifically recognize the extracellular domain (ECD) of human CAIX. Here we describe the biophysical and functional properties of a set of antibodies against the CAIX ECD domain and their applicability as: 1) suitable for development as an antibody-drug-conjugate, 2) an inhibitor of CAIX enzyme activity, or 3) an imaging/detection antibody. The results presented here demonstrate the potential of these specific hCAIX mAbs for further development as novel cancer therapeutic and/or diagnostic tools.

Summary Activating KRAS mutations are found in over 90% of pancreatic ductal adenocarcinomas (PDACs), yet KRAS has remained a difficult target to inhibit pharmacologically. Here, we demonstrate, using several human and mouse models of PDACs, rapid acquisition of tumor resistance in response to targeting KRAS or MEK, associated with integrin-linked kinase (ILK)-mediated increased phosphorylation of the mTORC2 component Rictor, and AKT. Although inhibition of mTORC1/2 results in a compensatory increase in ERK phosphorylation, combinatorial treatment of PDAC cells with either KRAS (G12C) or MEK inhibitors, together with mTORC1/2 inhibitors, results in synergistic cytotoxicity and cell death reflected by inhibition of pERK and pRictor/pAKT and of downstream regulators of protein synthesis and cell survival. Relative to single agents alone, this combination leads to durable inhibition of tumor growth and metastatic progression in vivo and increased survival. We have identified an effective combinatorial treatment strategy using clinically viable inhibitors, which can be applied to PDAC tumors with different KRAS mutations., Graphical Abstract, Highlights Inhibition of mutant KRAS or MEK leads to adaptive resistance in PDAC Mutant KRAS or MEK inhibition results in ILK-Rictor-mediated activation of AKT Targeting mutant KRAS or MEK and mTORC1/2 reduces growth and increases cytotoxicity Combination treatment sustains suppression of PDAC tumor progression in mice, The majority of pancreatic ductal adenocarcinomas are driven by mutant KRAS, yet targeting the canonical KRAS-MEK pathway has limited clinical impact. Brown et al. identify adaptive resistance mechanisms to mutant KRAS and MEK inhibitors and show that co-targeting mTORC1/2 leads to sustained suppression of tumor progression, revealing effective treatment strategies.

Invasion of neighboring extracellular matrix (ECM) by malignant tumor cells is a hallmark of metastatic progression. This invasion can be mediated by subcellular structures known as invadopodia, the function of which depends upon soluble N-ethylmaleimide-sensitive factor-activating protein receptor (SNARE)-mediated vesicular transport of cellular cargo. Recently, it has been shown the SNARE Syntaxin4 (Stx4) mediates trafficking of membrane type 1–matrix metalloproteinase (MT1-MMP) to invadopodia, and that Stx4 is regulated by Munc18c in this context. Here, it is observed that expression of a construct derived from the N-terminus of Stx4, which interferes with Stx4-Munc18c interaction, leads to perturbed trafficking of MT1-MMP, and reduced invadopodium-based invasion in vitro, in models of triple-negative breast cancer (TNBC). Expression of Stx4 N-terminus also led to increased survival and markedly reduced metastatic burden in multiple TNBC models in vivo. The findings are the first demonstration that disrupting Stx4-Munc18c interaction can dramatically alter metastatic progression in vivo, and suggest that this interaction warrants further investigation as a potential therapeutic target. Implications: Disrupting the interaction of Syntaxin4 and Munc18c may be a useful approach to perturb trafficking of MT1-MMP and reduce metastatic potential of breast cancers.

Solid tumors are characterized by poor vasculature, thereby causing insufficient oxygen and nutrient supply within the tumor. To thrive in such conditions, hypoxic tumor cells stimulate the expression of nutrient transporters to support cell metabolism and pH regulators to buffer the intracellular pH changes caused by metabolic by-products. Carbonic Anhydrase IX (CAIX) is a membrane-bound enzyme that plays a vital role in the pH regulation of hypoxic tumor cells. CAIX is highly expressed in several solid tumors, and is an indicator of poor prognosis and response to therapy. Although the importance of CAIX's role in mediating tumor progression is well-known, the underlying mechanism remains unclear. To identify the interactors of CAIX in the hypoxic tumor microenvironment, we performed a proteomic analysis using proximity dependent biotin identification (BioID) and found the Alanaine, Serine, Cysteine transporter (ASCT2) to be a high confidence interactor of CAIX from this analysis. ASCT2, which predominantly acts as a glutamine transporter is upregulated in several cancers and is shown to support tumor growth by facilitating increased glutamine uptake. In this study, using a panel of solid tumor cell lines, we validate the interaction of CAIX with ASCT2 in the hypoxic microenvironment. Functionally, loss of CAIX causes an increased glutamine uptake via ASCT2 and an increased capacity of cells to utilize glutamine for mitochondrial respiration. Concomitantly, ROS and glutathione (GSH) levels also increase upon CAIX inhibition. Cytotoxicity studies show a robust cell death in the absence of glutamine, following treatment with the CAIX/XII small molecule inhibitor SLC-0111. A similar effect on cell death is observed by blocking glutamine metabolism using inhibitors such as V-9302 (ASCT2 inhibitor) or CB-839 (Glutaminase-1 inhibitor) with SLC-0111. Furthermore, blocking GSH synthesis using BSO (Glutamylcysteine synthatase inhibitor) with SLC-0111 causes significant cell death. These results suggest that CAIX depletion causes increased cellular stress and to overcome this, cells increase glutamine uptake in part for GSH synthesis. In summary, we show that CAIX and ASCT2 coordinate their functions in hypoxic cancer cells to support tumor growth, and co-targeting them could be beneficial in treating solid tumors. Citation Format: Geetha Venkateswaran, Shawn Chafe, Shannon Awrey, Oksana Nemirovsky, Shoukat Dedhar. Interplay of the pH regulator, carbonic anhydrase IX and the glutamine transporter, ASCT2 in hypoxic 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 2015.

// Zhengcheng He 1,* , Nagarajan Kannan 2,3,* Oksana Nemirovsky 1 , Helen Chen 1 , Marisa Connell 1 , Brian Taylor 4 , Jihong Jiang 1 , Linda M. Pilarski 4 , Markus C. Fleisch 5 , Dieter Niederacher 6 , Miguel Angel Pujana 7 , Connie J. Eaves 2,8 and Christopher A. Maxwell 1,9 1 Department of Pediatrics, University of British Columbia, Vancouver, British Columbia, Canada 2 Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada 3 Department of Laboratory Medicine and Pathology, Division of Experimental Pathology and Laboratory Medicine, Mayo Clinic, Rochester, MN, USA 4 Department of Oncology, University of Alberta and Cross Cancer Institute, Edmonton, Alberta, Canada 5 Department of Obstetrics and Gynaecology, Landesfrauenklinik, HELIOS University Medical Center, Wuppertal, Germany 6 Department of Gynaecology and Obstetrics, University Hospital Dusseldorf, Heinrich-Heine University Dusseldorf, Germany 7 Breast Cancer and Systems Biology Unit, Program Against Cancer Therapeutic Resistance (ProCure), Catalan Institute of Oncology, IDIBELL, L’Hospitalet del Llobregat, Barcelona, Spain 8 Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada 9 Michael Cuccione Childhood Cancer Research Program, BC Children’s Hospital, Vancouver, British Columbia, Canada * These authors have contributed equally to this work Correspondence to: Christopher A. Maxwell, email: // Connie J. Eaves, email: // Keywords : BRCA1, spindle orientation, mitotic instability, human mammary epithelial cells Received : February 08, 2017 Accepted : February 16, 2017 Published : February 25, 2017 Abstract BRCA1 deficiency may perturb the differentiation hierarchy present in the normal mammary gland and is associated with the genesis of breast cancers that are genomically unstable and typically display a basal-like transcriptome. Oriented cell division is a mechanism known to regulate cell fates and to restrict tumor formation. We now show that the cell division axis is altered following shRNA-mediated BRCA1 depletion in immortalized but non-tumorigenic, or freshly isolated normal human mammary cells with graded consequences in progeny cells that include aneuploidy, perturbation of cell polarity in spheroid cultures, and a selective loss of cells with luminal features. BRCA1 depletion stabilizes HMMR abundance and disrupts cortical asymmetry of NUMA-dynein complexes in dividing cells such that polarity cues provided by cell-matrix adhesions were not able to orient division. We also show that immortalized mammary cells carrying a mutant BRCA1 allele ( BRCA1 185delAG/+ ) reproduce many of these effects but in this model, oriented divisions were maintained through cues provided by CDH1 + cell-cell junctions. These findings reveal a previously unknown effect of BRCA1 suppression on mechanisms that regulate the cell division axis in proliferating, non-transformed human mammary epithelial cells and consequent downstream effects on the mitotic integrity and phenotype control of their progeny.

Triple Negative Breast Cancer (TNBC) is aggressive, metastatic and drug-resistant, limiting the spectrum of effective therapeutic options for breast cancer patients. To date, anti-angiogenic agents have had limited success in the treatment of systemic breast cancer, possibly due to the exacerbation of tumor hypoxia and increased metastasis. Hypoxia drives increased expression of downstream effectors, including Carbonic Anhydrase IX (CAIX), a critical functional component of the pro-survival machinery required by hypoxic tumor cells. Here, we used the highly metastatic, CAIX-positive MDA-MB-231 LM2-4 orthotopic model of TNBC to investigate whether combinatorial targeting of CAIX and angiogenesis impacts tumor growth and metastasis in vivo to improve efficacy. The administration of a small molecule inhibitor of CAIX, SLC-0111, significantly reduced overall metastatic burden, whereas exposure to sunitinib increased hypoxia and CAIX expression in primary tumors, and failed to inhibit metastasis. The administration of SLC-0111 significantly decreased primary tumor vascular density and permeability, and reduced metastasis to the lung and liver. Furthermore, combining sunitinib and SLC-0111 significantly reduced both primary tumor growth and sunitinib-induced metastasis to the lung. Our findings suggest that targeting angiogenesis and hypoxia effectors in combination holds promise as a novel rational strategy for the effective treatment of patients with TNBC. Funding Agencies|Swedish Research Council (Vetenskapsradet)Swedish Research Council [537-2013-7467]; Canadian Institutes of Health Research (CIHR)Canadian Institutes of Health Research (CIHR) [143318]; Canadian Cancer Society Research Institute (CCSRI) [703191]

Treatment strategies involving immune-checkpoint blockade (ICB) have significantly improved survival for a subset of patients across a broad spectrum of advanced solid cancers. Despite this, considerable room for improving response rates remains. The tumor microenvironment (TME) is a hurdle to immune function, as the altered metabolism-related acidic microenvironment of solid tumors decreases immune activity. Here, we determined that expression of the hypoxia-induced, cell-surface pH regulatory enzyme carbonic anhydrase IX (CAIX) is associated with worse overall survival in a cohort of 449 patients with melanoma. We found that targeting CAIX with the small-molecule SLC-0111 reduced glycolytic metabolism of tumor cells and extracellular acidification, resulting in increased immune cell killing. SLC-0111 treatment in combination with immune-checkpoint inhibitors led to the sensitization of tumors to ICB, which led to an enhanced Th1 response, decreased tumor growth, and reduced metastasis. We identified that increased expression of CA9 is associated with a reduced Th1 response in metastatic melanoma and basal-like breast cancer TCGA cohorts. These data suggest that targeting CAIX in the TME in combination with ICB is a potential therapeutic strategy for enhancing response and survival in patients with hypoxic solid malignancies.

Activating mutations in the KRAS oncogene are found in greater than 90% of pancreatic ductal adenocarcinoma (PDAC), yet KRAS remains difficult to inhibit pharmacologically. Small molecule inhibitors of RAF/MEK/ERK (MAPK) or mTOR are first line treatments for several human malignancies, however resistance to these targeted therapies continues to be a major clinical problem. We show in vitro dose dependent inhibition of proliferation and pERK1/2 in human PDAC cell lines and a KRAS-driven genetically engineered mouse model (GEMM) of pancreatic cancer in response to Trametinib, a small molecule MEK-inhibitor (MEKi). Similarly, Trametinib elicited a dose dependent inhibition of the in vivo tumor growth of a subcutaneous human PDAC xenograft, however these tumors rapidly developed resistance and contained areas of increased activation of pAkt(Ser473). We determined that genetic depletion of KRAS using doxycycline-inducible shRNA or small molecule MEKi leads to increased pAkt(Ser473) and activation of mTORC2 by western blot, implicating activation of the Akt/mTORC1/2 pathway may be a mechanism of resistance to MEKi. Interestingly, treatment of PDAC cell lines with Torin1, a small molecule inhibitor of mTORC1/2, results in the increased activation of pERK1/2, suggesting crosstalk and compensation of these two pathways as mechanisms of resistance. The combination of Trametinib and Torin1 leads to the inhibition of both pERK1/2 and pAkt(Ser473) that is accompanied by a dose dependent, synergistic increase in cytotoxicity not observed with either single agent. In human PDAC xenografts and a syngeneic GEMM, combination treatment of Trametinib and Torin1 dramatically reduced tumor burden and increased survival, without serious toxicity. These results demonstrate that combined inhibition of MEK/ERK and mTORC1/2 is a potent and viable therapeutic strategy for PDAC. Citation Format: Wells Brown, Oksana Nemirovsky, Jordan Gillespie, Paul McDonald, Shoukat Dedhar. Combination MEK and mTORC1/2 inhibition overcomes resistance to single agent therapy in pancreatic cancer [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 1906.

Construction of a mitotic spindle requires biochemical pathways to assemble spindle microtubules and structural proteins to organize these microtubules into a bipolar array. Through a complex with dynein, the receptor for hyaluronan-mediated motility (RHAMM) cross-links mitotic microtubules to provide structural support, maintain spindle integrity, and correctly orient the mitotic spindle. Here, we locate RHAMM to sites of microtubule assembly at centrosomes and non-centrosome sites near kinetochores and demonstrate that RHAMM is required for the activation of Aurora kinase A. Silencing of RHAMM delays the kinetics of spindle assembly, mislocalizes targeting protein for XKlp2 (TPX2), and attenuates the localized activation of Aurora kinase A with a consequent reduction in mitotic spindle length. The RHAMM-TPX2 complex requires a C-terminal basic leucine zipper in RHAMM and a domain that includes the nuclear localization signal in TPX2. Together, our findings identify RHAMM as a critical regulator for Aurora kinase A signaling and suggest that RHAMM ensures bipolar spindle assembly and mitotic progression through the integration of biochemical and structural pathways.

Malignant peripheral nerve sheath tumours (MPNST) are rare, hereditary cancers associated with neurofibromatosis type I. MPNSTs lack effective treatment options as they often resist chemotherapies and have high rates of disease recurrence. Aurora kinase A (AURKA) is an emerging target in cancer and an aurora kinase inhibitor (AKI), termed MLN8237, shows promise against MPNST cell lines in vitro and in vivo. Here, we test MLN8237 against two primary human MPNST grown in vivo as xenotransplants and find that treatment results in tumour cells exiting the cell cycle and undergoing endoreduplication, which cumulates in stabilized disease. Targeted therapies can often fail in the clinic due to insufficient knowledge about factors that determine tumour susceptibilities, so we turned to three MPNST cell-lines to further study and modulate the cellular responses to AKI. We find that the sensitivity of cell-lines with amplification of AURKA depends upon the activity of the kinase, which correlates with the expression of the regulatory gene products TPX2 and HMMR/RHAMM. Silencing of HMMR/RHAMM, but not TPX2, augments AURKA activity and sensitizes MPNST cells to AKI. Furthermore, we find that AURKA activity is critical to the propagation and self-renewal of sphere-enriched MPNST cancer stem-like cells. AKI treatment significantly reduces the formation of spheroids, attenuates the self-renewal of spheroid forming cells, and promotes their differentiation. Moreover, silencing of HMMR/RHAMM is sufficient to endow MPNST cells with an ability to form and maintain sphere culture. Collectively, our data indicate that AURKA is a rationale therapeutic target for MPNST and tumour cell responses to AKI, which include differentiation, are modulated by the abundance of HMMR/RHAMM.

Reactive oxygen species (ROS), a byproduct of cellular metabolism, damage intracellular macromolecules and, when present in excess, can promote normal hematopoietic stem cell differentiation and exhaustion. However, mechanisms that regulate the amount of ROS in leukemia-initiating cells (LICs) and the biological role of ROS in these cells are largely unknown. We show here that the ROS(low) subset of CD44(+) cells in T cell acute lymphoblastic leukemia (T-ALL), a malignancy of immature T cell progenitors, is highly enriched in the most aggressive LICs and that ROS accumulation is restrained by downregulation of protein kinase C θ (PKC-θ). Notably, primary mouse T-ALLs lacking PKC-θ show improved LIC activity, whereas enforced PKC-θ expression in both mouse and human primary T-ALLs compromised LIC activity. We also show that PKC-θ is regulated by a new pathway in which NOTCH1 induces runt-related transcription factor 3 (RUNX3), RUNX3 represses RUNX1 and RUNX1 induces PKC-θ. NOTCH1, which is frequently activated by mutation in T-ALL and required for LIC activity in both mouse and human models, thus acts to repress PKC-θ. These results reveal key functional roles for PKC-θ and ROS in T-ALL and suggest that aggressive biological behavior in vivo could be limited by therapeutic strategies that promote PKC-θ expression or activity, or the accumulation of ROS.

Mucopolysaccharidosis III (MPS III) has four forms (A–D) that result from buildup of an improperly degraded glycosaminoglycan in lysosomes. MPS IIIB is attributable to the decreased activity of a lysosomal α- N -acetylglucosaminidase (NAGLU). Here, we describe the structure, catalytic mechanism, and inhibition of Cp GH89 from Clostridium perfringens , a close bacterial homolog of NAGLU. The structure enables the generation of a homology model of NAGLU, an enzyme that has resisted structural studies despite having been studied for >20 years. This model reveals which mutations giving rise to MPS IIIB map to the active site and which map to regions distant from the active site. The identification of potent inhibitors of Cp GH89 and the structures of these inhibitors in complex with the enzyme suggest small-molecule candidates for use as chemical chaperones. These studies therefore illuminate the genetic basis of MPS IIIB, provide a clear biochemical rationale for the necessary sequential action of heparan-degrading enzymes, and open the door to the design and optimization of chemical chaperones for treating MPS IIIB.

PROBLEM- Metastasis accounts for 90% of deaths from carcinomas. BACKGROUND- To initiate migration, epithelial cells must front-rear polarize and reorganize the microtubule cytoskeleton directed toward the lamillipodia or leading edge. During mitosis, centrosome maturation, duplication, spindle pole assembly and chromosome segregation require Aurora kinase A (AURKA), which is optimally active when complexed with TPX2. Accumulating evidences identify non-mitotic functions for AURKA, such as cell migration and ciliary reorganization. Overexpression of AURKA leads to tumorigenesis in breast cancer. Mechanisms that activate non-mitotic AURKA and promote cell migration as well as metastasis are yet to be uncovered. RESULTS- In a large breast cancer tissue microarray (n= 3,175), the abundance of phosphorylated RHAMM (a substrate of AURKA) predicted overall and relapse-free survival in ER-negative, basal-subtype, and triple negative breast tumors. We studied the AURKA-TPX2-RHAMM signaling axis in mammary (MCF10A and nMUMG-FUCCI) and HeLa cells. In scratch wound assays, migratory cells were found to have front-polarized centrosomes, which correlated with G2 cell cycle phase, phosphorylated AURKA and elevated microtubule nucleation at centrosomes. Small molecule inhibition of Aurora kinase activity impaired centrosome polarity and retarded the kinetics of cell migration. In parallel, silencing RHAMM altered TPX2 location as indicated by immunofluorescence and immuno-EM, which located TPX2 to the nuclear envelope and nuclear pore basket. Nuclear import of recombinant TPX2 was attenuated in RHAMM silenced cells and nuclear envelope accumulation was elevated. CONCLUSION- The AURKA-TPX2-RHAMM axis regulates centrosome polarity, microtubule nucleation and directional cell migration while phosphorylated RHAMM predicts survival in aggressive forms of breast cancer. Citation Format: Tony Lok Heng Chu, Lixin Zhou, Jennifer Won, Pooja Mohan, Oksana Nemirovsky, Abbas Fotovati, Torsten Nielsen, Nelly Pante, Christopher Maxwell. Cell cycle-dependent front polarized cell migration requires Aurora kinase A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1876. doi:10.1158/1538-7445.AM2017-1876

BRCA1 deficiency alters the relative proportions of progenitor cells in preneoplastic mammary tissue, and typically associates with breast cancers characterized by genomic instability and a basal-like cell phenotype. Oriented division of progenitor cells is one mechanism these cells use to maintain tissue homeostasis, and to suppress tumor formation. We now show that shRNA-mediated reduction of BRCA1 levels in non-tumorigenic and immortalized or freshly isolated, normal human mammary cells alters their plane of division with graded consequences that include the induction of aneuploidy in progeny cells, perturbation of polarity in spheroid cultures, and inhibition of clonal growth with favored expression of basal features. We also demonstrate a requirement for BRCA1 in establishing cortical asymmetry of NUMA-dynein complexes. Mutation of a single BRCA1 allele (BRCA1 185delAG/+) altered the division axis of isolated cells but their deficient spindle positioning was supervised by CDH1-positive adherens, which sustained oriented divisions and produced colonies with luminal features. These findings reveal a previously unrecognized consequence of mutant BRCA1 on the cell division axis, post-mitotic integrity and phenotype control in normal human mammary epithelial cells. Note: This abstract was not presented at the meeting. Citation Format: Zhengcheng He, Oksana Nemirovsky, Nagarajan Kannan, Connie Eaves, Christopher A. Maxwell. BRCA1 controls the cell division axis and governs ploidy and phenotype in human mammary cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3459. doi:10.1158/1538-7445.AM2017-3459

International audience; Remnants of ancient transposable elements (TEs) are abundant in mammalian genomes. These sequences harbor multiple regulatory motifs and hence are capable of influencing expression of host genes. In response to environmental changes, TEs are known to be released from epigenetic repression and to become transcriptionally active. Such activation could also lead to lineage-inappropriate activation of oncogenes, as one study described in Hodgkin lymphoma. However, little further evidence for this mechanism in other cancers has been reported. Here, we reanalyzed whole transcriptome data from a large cohort of patients with diffuse large B-cell lymphoma (DLBCL) compared with normal B-cell centroblasts to detect genes ectopically expressed through activation of TE promoters. We have identified 98 such TE-gene chimeric transcripts that were exclusively expressed in primary DLBCL cases and confirmed several in DLBCL-derived cell lines. We further characterized a TE-gene chimeric transcript involving a fatty acid-binding protein gene (LTR2-FABP7), normally expressed in brain, that was ectopically expressed in a subset of DLBCL patients through the use of an endogenous retroviral LTR promoter of the LTR2 family. The LTR2-FABP7 chimeric transcript encodes a novel chimeric isoform of the protein with characteristics distinct from native FABP7. In vitro studies reveal a dependency for DLBCL cell line proliferation and growth on LTR2-FABP7 chimeric protein expression. Taken together, these data demonstrate the significance of TEs as regulators of aberrant gene expression in cancer and suggest that LTR2-FABP7 may contribute to the pathogenesis of DLBCL in a subgroup of patients.

Cell cycle progression and the acquisition of a migratory phenotype are hallmarks of human carcinoma cells. Here we report a strong correlation between cell cycle progression into G2 phase and an increased migratory velocity for non-malignant, immortalized mammary epithelia nMuMG (Mus musculus mammary gland) cells, or malignant cervical carcinoma HeLa cells, expressing a fluorescent ubiquitin cell cycle indicator and quantified in wound closure assays with high content multi-parameter live cell imaging. Cells at the wound edge exhibited elevated microtubule nucleation capacity at centrosomes, and a reduction of this capacity through inhibition of aurora kinase A was sufficient to impair migration velocity without affecting cell cycle dynamics. In migratory cells, active aurora kinase A at the centrosome correlated with nucleation capacity. In clinically annotated mammary carcinoma tissues (n=3,992), aurora kinase A activity, as assessed by the abundance of phosphorylated-RHAMM (Receptor for Hyaluronan Mediated Motility) was a significant predictor of breast cancer specific survival and relapse free survival in patients with estrogen receptor negative breast cancer (n= 941), triple negative phenotype breast cancer (n= 538) or basal-like subtype breast cancer (n= 293), but not in those patients with estrogen receptor positive breast cancer (n= 2,218). In estrogen receptor negative tumors from patients that received no adjuvant systemic therapy (n= 453), the levels of phosphorylated-RHAMM associated with the presence of distal nodal metastasis and predicted a subset of patients at high risk of recurrence. Together, these data identify aurora kinase A activity as a regulatory pathway that intersects cell cycle progression and cell migration, with potential to influence the metastasis of estrogen receptor negative breast tumors and patient survival. Citation Format: Tony LH Chu, Jennifer Won, Oksana Nemirovsky, Abbas Fotovati, Torsten Nielsen, Christopher A Maxwell. Cell cycle regulated centrosome orientation during directed migration requires Aurora Kinase A. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A61.

Malignant peripheral nerve sheath tumours (MPNST) are rare, hereditary, cancers associated with mutations in the neurofibromin 1 gene 1. MPNSTs are often resistant to chemotherapies and have high rates of disease recurrence, highlighting the lack of effective treatment options for this cancer. Aurora kinase A inhibitors (AKIs) have shown promise against MPNST cell lines 2. We expanded this study by testing AKI in human MPNST xenotransplant mice models. Treatment resulted in stabilized disease with tumor cells undergoing senescence and endoreduplication. Aurora kinase A (AURKA) is an emerging target in cancer, however, targeted therapies can often fail in the clinic due to insufficient knowledge about factors that determine tumor response. Therefore, we utilized three MPNST cell lines and profiled them for the expression and activity of AURKA as well as their responses to AKIs. The most proliferative lines, S462 and 2884, express equivalent levels of AURKA, however, S462 cells were more sensitive to kinase inhibition. Both cell lines experienced apoptosis, senescence and endoreduplication in response to AKI treatment. AURKA activity is regulated by a co-activator, the Targeting Protein for XKlp2 (TPX2) and a molecular brake, the Receptor for Hyaluronan Mediated Motility (RHAMM)3. Interestingly, published analysis of copy number variation has identified hemizygous loss of the RHAMM gene in half of the examined high-grade MPNST, but not in benign or low grade tumors 4. We proposed that MPNSTs with RHAMM deletions are oncogene addicted to AURKA activity and are therefore, particularly susceptible to AKI. We profiled our MPNST lines for RHAMM and TPX2 expression and found that S462 cells express significantly more TPX2 and less RHAMM compared to 2884 cells. Furthermore, S462 cells had increased kinase. To determine whether levels of these molecular regulators could affect AKI efficacy we depleted RHAMM and TPX2 in 2884 and S462 cells respectively. While cells with reduced TPX2 have unchanged responses to AKIs, RHAMM depleted cells have a 2 fold reduction in IC-50s. We also looked at the effect of AKI against a population of MPNST tumor-initiating cells (TICs) from the S462 line. Compared to adherent cells, S462 TICs have elevated AURKA activity and their ability to self-renew in vitro is arrested by AKI. Indeed, the altered levels of kinase activity in the RHAMM and TPX2 depletion lines correlated with their ability to form and maintain sphere culture. In addition, we find that AKI treated S462 TICs differentiated into terminal neurons. All in all, these data indicate AURKA as a rational therapy for aggressive MPNSTs with RHAMM serving as a biomarker for AKI efficacy. Citation Format: Pooja Mohan, Joan Castellsague, Jihong Jiang, Kristi Allen, Helen Chen, Oksana Nemirovsky, Melanie Spyra, Kaiji Hu, Lan Kluwe, Miguel Pujana, Alberto Villanueva, Victor Mautner, Sandra Dunn, Jonathan Keats, Conxi Lazaro, Christopher Maxwell. Common genomic alterations in malignant peripheral nerve sheath tumors augment Aurora A activity and sensitize tumors to aurora kinase inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-214. doi:10.1158/1538-7445.AM2013-LB-214

Notch-driven expression of IGF1R promotes the growth, viability, and transplantability of T-ALL cells., T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer of immature T cells that often shows aberrant activation of Notch1 and PI3K–Akt pathways. Although mutations that activate PI3K–Akt signaling have previously been identified, the relative contribution of growth factor-dependent activation is unclear. We show here that pharmacologic inhibition or genetic deletion of insulin-like growth factor 1 receptor (IGF1R) blocks the growth and viability of T-ALL cells, whereas moderate diminution of IGF1R signaling compromises leukemia-initiating cell (LIC) activity as defined by transplantability in syngeneic/congenic secondary recipients. Furthermore, IGF1R is a Notch1 target, and Notch1 signaling is required to maintain IGF1R expression at high levels in T-ALL cells. These findings suggest effects of Notch on LIC activity may be mediated in part by enhancing the responsiveness of T-ALL cells to ambient growth factors, and provide strong rationale for use of IGF1R inhibitors to improve initial response to therapy and to achieve long-term cure of patients with T-ALL.

Abstract 12 T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T cell progenitors which affects both children and adults. Current therapies achieve cure in about 80% of pediatric patients, but only 40% of adults survive beyond 5 years. Disease relapse may be attributed to ineffective targeting of leukemia stem cells (LSCs) due to their presumed quiescence, resistance to apoptosis, enhanced expression of DNA repair enzymes and drug efflux pumps, and localization to protective or inaccessible niches. More efficient targeting of LSCs in this disease thus could offer great potential for improving patient outcomes. Recent interest in the role of reactive oxygen species (ROS) in cancer stem cell biology has been sparked by studies demonstrating that low ROS levels promote clonogenicity and radioresistance of breast cancer stem cells and that parthenolide specifically induces apoptosis of LSCs in AML and blast crisis CML by increasing levels of ROS in these cells. In T-ALL, activation of NOTCH1 signaling, either by mutation of NOTCH1 itself (50-60% of cases) or its negative regulator FBW7 (15% of cases), contributes to malignant behavior by blocking differentiation, promoting cellular proliferation/survival, and enhancing self-renewal. Similar gain-of-function Notch1 mutations are also observed to occur spontaneously in several murine T-ALL models at high frequency. To examine ROS levels in T-ALL LSCs, we first generated murine disease by retroviral transduction of bone marrow with a constitutively activated form of NOTCH1 termed “ΔE”. We utilized limiting dilution transplantation analyses of different cellular phenotypes isolated from primary ΔE leukemias to demonstrate that the CD44+ subset of CD4+ CD8+ (DP) cells were enriched for LSCs and exhibited lower ROS levels as compared to the CD44- DP subset and bulk cells. Since ROS levels were heterogeneous within the CD44+ DP subset, we combined these markers with DC-FDA staining for ROS and demonstrated as few as 100 ROSlow CD44+ DP cells are able to reproduce disease in 100% of recipients whereas 100,000 bulk cells are required to achieve the same effect. Because protein kinase C theta (PKCθ) was recently identified as an important mediator of ROS production in T-cells during activation-induced cell death and expression of PKCθ mRNA could be detected broadly in primary human and mouse T-ALL samples, we reasoned that this signaling intermediate might be a regulator of ROS levels in malignant T-ALL LSCs. In fact, using a combination of viral expression and knock-down approaches in both human and mouse T-ALLs as well as mouse PKCθnull ΔE leukemias, we demonstrate that PKCθ is a primary regulator ROS production in T-ALL. Moreover, low PKCθ/ROS levels correlate with resistance to chemotherapy and irradiation, and promote the competitive in vivo growth of T-ALL cells. Interestingly, we also find that Notch signaling represses PKCθ expression which in turn results in reduced ROS levels. Our results identify PKCθ as a novel target of Notch signaling in LSCs whose key malignant properties are highly dependent on low ROS levels. These findings provide mechanistic basis for rational design of LSC-targeted therapies in T-ALL. Disclosures: No relevant conflicts of interest to declare.

Abstract 3649 T-cell acute lymphoblastic leukemia (T-ALL) is a lethal cancer of immature T cells that often shows aberrant activation of NOTCH1 and PI3K/Akt pathways. Although PI3K/Akt signaling is potentiated by PTEN mutations which occur in up to 20% of cases, upstream signals which initiate PI3K/Akt activation remain unclear. IGF1R is a receptor tyrosine kinase upstream of PI3K/Akt and Ras/Raf/MAPK signaling pathways with known roles in cancer cell growth and is expressed broadly in primary human T-ALL cells and established cell lines. We thus considered that IGF1R may represent a biologically relevant upstream activator of the PI3K/Akt pathway in T-ALL. Consistent with this idea, we demonstrate that treatment of human and mouse T-ALL cells with small molecule IGF1R kinase inhibitors and IGF1R blocking antibodies resulted in growth arrest of bulk cells. As well, genetic deletion of IGF1R by inducible Cre expression in mouse T-cell leukemias also leads to growth arrest and apoptosis of bulk cells. IGF1R and PI3K/Akt/mTOR signaling have also been described to promote self-renewal of normal and cancer stem cells, and thus we addressed whether IGF1R may play a role in leukemia stem cell (LSC) activity in T-ALL. Since outgrowth of bulk leukemia cells is required to detect LSC activity in vivo, we were not able to address IGF1R function in LSCs by genetic deletion; however, we elected to take advantage of a well-characterized hypomorphic allele of IGF1R carrying an integrated neo cassette within the second intron. IGF1Rneo/neo mice express full-length IGF1R protein, but at reduced levels (30-60% of wild-type). Despite this reduced expression level, we were able to generate lethal T-cell leukemias with 100% penetrance in primary recipients of IGF1Rneo/neo bone marrow transduced with activated NOTCH1 retrovirus, similar to what is observed with IGF1R+/+ bone marrow. Upon IV or intra-medullary injection into secondary congenic recipients, however, IGF1Rneo/neo leukemias exhibited a striking transplantation defect. Whereas IGF1R+/+ leukemias are consistently serially transplantable, 5 of 16 primary IGF1Rneo/neo leukemias failed to reconstitute disease in any secondary recipients, 5 produced disease in only a subset of secondary recipients, and 6 produced disease in all secondary recipients. Furthermore, treatment of IGF1R+/+ leukemia cells in vitro with IGF1R inhibitor prior to injection into secondary recipients prevented their transplantability, whereas treatment with PI3K and ERK inhibitors did not. Finally, given that inhibition of Notch signaling in human T-ALL cells compromises their ability to recapitulate disease in NOD/Scid recipients, we wondered whether this effect could be mediated by downregulation of IGF1R. In fact, we observed inhibition of Notch signaling, either by small molecule gamma-secretase inhibitor or transduction with dominant negative Mastermind, to result in a 2–3 fold decrease in IGF1R protein expression, corresponding to a 5 to 20-fold decrease in response to stimulation by IGF-1 ligand. Thus, these studies demonstrate high-level IGF1R signaling is required for LSC activity in T-ALL, and that Notch signaling may promote LSC function by supporting IGF1R expression. IGF1R inhibitors may therefore prove clinically useful in preventing disease relapse by compromising LSC self-renewal. Disclosures: Carboni: Bristol-Myers Squibb: Employment. Gottardis:Bristol-Myers Squibb: Employment.