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Ectopic expression of CAAT/enhancer binding protein α (C/EBPα) in p210BCR/ABL-expressing cells induces granulocytic differentiation, inhibits proliferation, and suppresses leukemogenesis. To dissect the molecular mechanisms underlying these biological effects, C/EBPα-regulated genes were identified by microarray analysis in 32D-p210BCR/ABL cells. One of the genes whose expression was activated by C/EBPα in a DNA binding–dependent manner in BCR/ABL-expressing cells is the transcriptional repressor Gfi-1. We show here that C/EBPα interacts with a functional C/EBP binding site in the Gfi-1 5′-flanking region and enhances the promoter activity of Gfi-1. Moreover, in K562 cells, RNA interference–mediated downregulation of Gfi-1 expression partially rescued the proliferation-inhibitory but not the differentiation-inducing effect of C/EBPα. Ectopic expression of wild-type Gfi-1, but not of a transcriptional repressor mutant (Gfi-1P2A), inhibited proliferation and markedly suppressed colony formation but did not induce granulocytic differentiation of BCR/ABL-expressing cells. By contrast, Gfi-1 short hairpin RNA–tranduced CD34+ chronic myeloid leukemia cells were markedly more clonogenic than the scramble-transduced counterpart. Together, these studies indicate that Gfi-1 is a direct target of C/EBPα required for its proliferation and survival-inhibitory effects in BCR/ABL-expressing cells. Cancer Res; 70(20); 7949–59. ©2010 AACR.

Supplementary Figure 1 from Expression of the Transcriptional Repressor Gfi-1 Is Regulated by C/EBPα and Is Involved in Its Proliferation and Colony Formation–Inhibitory Effects in p210BCR/ABL-Expressing Cells

Despite considerable progress made in improving therapeutic strategies, the overall survival for patients diagnosed with various cancer types remains low. Further, patients often cycle through multiple therapeutic options before finding an effective regimen for the specific malignancy being treated. A focus on building enhanced computational models, which prioritize therapeutic regimens based on a tumor’s complete molecular profile, will improve the patient experience and augment initial outcomes. In this study, we present an integrative analysis of multiple omic datasets coupled with phenotypic and therapeutic response profiles of Cytarabine from a cohort of primary AML tumors, and Olaparib from a cohort of Patient-Derived Xenograft (PDX) models of ovarian cancer. These analyses, termed Pharmaco-Pheno-Multiomic (PPMO) Integration, established novel complex biomarker profiles that were used to accurately predict prospective therapeutic response profiles in cohorts of newly profiled AML and ovarian tumors. Results from the computational analyses also provide new insights into disease etiology and the mechanisms of therapeutic resistance. Collectively, this study provides proof-of-concept in the use of PPMO to establish highly accurate predictive models of therapeutic response, and the power of leveraging this method to unveil cancer disease mechanisms.

The immune cell composition of isocitrate dehydrogenase wild type (IDH-wt) glioma patients significantly differs compared to IDH-mutant (IDH-mut) yet a detailed and unbiased understanding of their transcriptomic and epigenetic landscapes remains elusive. To this end, we performed single-cell RNA-sequencing (scRNA-seq) and single-cell Assay for Transposase-Accessible Chromatin using sequencing (sc-ATAC-seq) on ~100,000 tumor-associated immune cells from seventeen IDH mutation classified primary and recurrent human gliomas and non-glioma brains (NGBs). Our analyses revealed sixty-two transcriptionally distinct myeloid and lymphoid cell states within and across glioma subtypes and we noted microglial attrition with increasing disease severity concomitant with invading monocyte-derived cells (MDCs) and lymphocytes. Specifically, certain microglial and monocyte-derived subpopulations were associated with antigen presentation gene modules, akin to cross-presenting dendritic cells. As tissue macrophages exhibit multifaceted polarization in response to microenvironmental cues, we clarify the existence of microglia/macrophage functional states beyond M1/M2 paradigms exemplified by the presence of palmitic-, oleic- acid, and glucocorticoid responsive polarized states. We identified cytotoxic T cells with poly-functional cytolytic states mostly in recurrent IDH-wt gliomas. Furthermore, ligand-receptor interactome analyses showed a preponderance of antigen presentation/phagocytosis over the checkpoint axis in IDH-wt compared to IDH-mut gliomas. Additionally, our sc-ATAC-seq analyses revealed differences in regulatory networks in NGBs, IDH-mut, and IDH-wt glioma-associated immune cells. In particular, we noted reduced microglial usage of an iron recycling SPIC transcription factor and Interferon Regulatory Factors (IRFs); IRF1 and IRF2 in IDH-wt relative to IDH-mut and NGBs. Unique features such as amplification of 11-Zinc Finger Protein accessibility were restricted to MDCs. Finally, sc-ATAC-seq profiles of CD8+ exhausted T cells from IDH-wt showed strong enhancer accessibility on CTLA-4, Layilin, and TIM-3 but no enrichment on PD1 was seen. In summary, our study provides unprecedented granular detail of transcriptionally and epigenomically defined glioma-specific immune contexture that can be exploited for immunotherapy applications.

Introduction: Immunotherapies are focused on strategies that alter immune responses, using antibodies that binds to receptors on different immune cell subsets and either activate or suppress their functions depending on the immune response being targeted. Hence, the necessity of developing assays that assess the functional and biological effect of a therapeutic on its target. When incorporated into high-parameter flow cytometry panels, receptor occupancy assay can simultaneously evaluate receptor expression and drug occupancy on defined cell subsets, which can provide information related to functional effects, and safety.Areas covered: This review focuses on the importance of developing, optimizing, and validating a robust Receptor Occupancy Assay (ROA) to improve dose selection, pharmacology monitoring and safety mainly in clinical settings.Expert opinion: The designing of an ROA can be challenging and can lead to exaggerated pharmacology if not accurately developed, optimized, and validated. However, improvements in our understanding of epitopes, binding, affinities, and pharmacological effects may lead to improved antibody drug targeting and receptor evaluation.

The overall survival of patients diagnosed with Acute Myeloid Leukemia (AML) remains low. While initial responses to therapy are favorable, the duration of response is short and overcoming therapeutic resistance has proven difficult. A better understanding of the tumor cell biology and resistance mechanisms may shed light onto novel therapeutic targets that improve long-term outcome. In this study, we performed an exhaustive analysis to include deep tumor phenotyping, drug sensitivity profiling and comprehensive omic characterization. These datasets were included in integrative pharmaco-phenotypic-multiomic analyses to identify targets and biomarkers associated with cellular phenotype and drug response. Our results reveal that the major cellular discriminant within the cellular phenotype is CD34 expression, which associates with a high PDK-mediated metabolic profile and cytarabine sensitivity. Tumors exhibiting cytarabine resistance associate with a CD34-negative cellular phenotype and molecular characteristics such as MYC copy number gain, and increased expression of SAMDH1, FBP1 and TYMP proteins. Citation Format: Gilad Silberberg, Bandana Vishwakarama, Brandon Walling, Chelsea Riveley, Alessandra Audia, Marianna Zipeto, Ido Sloma, Amy Wesa, Michael Ritchie. A pheno-multiomic integration analysis of primary samples of acute myeloid leukemia reveals biomarkers of cytarabine resistance [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 3907.

The brain tumor immune microenvironment (TIME) continuously evolves during glioma progression, but a comprehensive characterization of the glioma-centric immune cell repertoire beyond a priori cell states is uncharted. In this study, we performed single-cell RNA-sequencing (scRNA-seq) and single cell- Assay for Transposase-Accessible Chromatin using sequencing (sc-ATAC-seq) on ~100,000 tumor-associated immune cells from seventeen isocitrate dehydrogenase (IDH) mutation classified primary and recurrent human gliomas and non-glioma brains (NGBs). Our analyses revealed sixty-two transcriptionally distinct myeloid and lymphoid cell states within and across glioma subtypes and we noted microglial attrition with increasing disease severity concomitant with invading monocyte-derived cells and lymphocytes. Specifically, certain microglial and monocyte-derived subpopulations were associated with antigen presentation gene modules, akin to cross-presenting dendritic cells (DCs). We identified cytotoxic T cells with poly-functional cytolytic states mostly in recurrent IDH-wt gliomas. Furthermore, ligand-receptor interactome analyses showed a preponderance of antigen presentation and phagocytosis over the checkpoint axis in IDH-wt compared to IDH-mut gliomas. Additionally, our sc-ATAC-seq analyses revealed differences in regulatory networks in NGBs, IDH-mut and IDH-wt glioma associated immune cells. In particular, we noted abundant usage of inflammatory transcription factors (TFs) as exemplified by Nuclear factor kappa B and Activator Protein-1 TF family in IDH-wt microglia when compared with microglia from IDH-mut and NGBs. Unique features such as amplification of 11- Zinc Finger Protein accessibility were restricted to monocyte derived cells and were not observed in microglia. Finally, sc-ATAC-seq profiles of CD8+ exhausted T cells from IDH-wt showed strong enhancer accessibility on Cytotoxic T-lymphocyte-associated protein 4, Layilin and Hepatitis A Virus Cellular Receptor 2 but no enrichment on PDCD1 (gene encoding Programmed cell death protein 1) was seen. In summary, our study provides unprecedented granular detail of transcriptionally defined glioma- specific immune contexture that can be exploited for immunotherapy applications. This study in K.B. laboratory was supported by the generous philanthropic contributions to The University of Texas (UT) MD Anderson Cancer Center (MDACC) Moon Shots Program™, Marnie Rose Foundation, NIH grants: R21 CA222992 and R01CA225963. This study was partly supported by the UT MDACC start-up research fund to L.W. and CPRIT Single Core grant RP180684 to N. E. N.

15 páginas, 7 figuras. Contiene información suplementaria en l aversión online., A mesenchymal transition occurs both during the natural evolution of glioblastoma (GBM) and in response to therapy. Here, we report that the adhesion G-protein-coupled receptor, GPR56/ADGRG1, inhibits GBM mesenchymal differentiation and radioresistance. GPR56 is enriched in proneural and classical GBMs and is lost during their transition toward a mesenchymal subtype. GPR56 loss of function promotes mesenchymal differentiation and radioresistance of glioma initiating cells both in vitro and in vivo. Accordingly, a low GPR56-associated signature is prognostic of a poor outcome in GBM patients even within non-G-CIMP GBMs. Mechanistically, we reveal GPR56 as an inhibitor of the nuclear factor kappa B (NF-κB) signaling pathway, thereby providing the rationale by which this receptor prevents mesenchymal differentiation and radioresistance. A pan-cancer analysis suggests that GPR56 might be an inhibitor of the mesenchymal transition across multiple tumor types beyond GBM., This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (BFU2009-14616 and RyC-09-389-10-01 to N.d.l.I.), Fundación Ramón Areces (CIVP16A1850 to N.d.l.I.), and an FPI predoctoral fellowship from MINECO (to M. Moreno). This research was also supported by NIH grant R01NS083767 (to I.N.) and the University Cancer Foundation via the Institutional Research Grant program and start-up funds at the University of Texas M.D. Anderson Cancer Center (to K.P.B.). This work was co-funded by the European Regional Development Fund (ERDF) and the CERCA Programme (Generalitat de Catalunya).

Summary Glioblastoma (GBM) is the most prevalent and aggressive tumor in the central nervous system. Surgical resection followed by concurrent radiotherapy (ionizing radiation [IR]) and temozolomide (TMZ) is the standard of care for GBM. However, a large subset of patients offer resistance or become adapted to TMZ due mainly to the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Thus, alternative mechanisms of MGMT deregulation have been proposed but are heretofore unproven. We show that heterogeneous GBM cells express tunneling nanotubes (TNTs) upon oxidative stress and TMZ/IR treatment. We identified that MGMT protein diffused from resistant to sensitive cells upon exposure to TMZ/IR, resulting in protection against cytotoxic therapy in a TNT-dependent manner. In vivo analysis of resected GBM tumors support our hypothesis that the MGMT protein, but not its mRNA, was associated with TNT biomarkers. We propose that targeting TNT formation could be an innovative strategy to overcome treatment resistance in GBM., Graphical Abstract, Highlights • GBM cells form TNTs in response to ionizing radiation and TMZ treatment • TNTs transfer MGMT protein from resistant to sensitive cells to treatment • MGMT protein transferred via TNTs protect target cells from TMZ and IR treatment • Blocking TNTs and MGMT spread could prevent tumor adaptation to TMZ treatment, Cell Biology; Cancer

Tumor microenvironments can promote stem cell maintenance, tumor growth, and therapeutic resistance, findings linked by the tumor-initiating cell hypothesis. Standard of care for glioblastoma (GBM) includes temozolomide chemotherapy, which is not curative, due, in part, to residual therapy-resistant brain tumor-initiating cells (BTICs). Temozolomide efficacy may be increased by targeting carbonic anhydrase 9 (CA9), a hypoxia-responsive gene important for maintaining the altered pH gradient of tumor cells. Using patient-derived GBM xenograft cells, we explored whether CA9 and CA12 inhibitor SLC-0111 could decrease GBM growth in combination with temozolomide or influence percentages of BTICs after chemotherapy. In multiple GBMs, SLC-0111 used concurrently with temozolomide reduced cell growth and induced cell cycle arrest via DNA damage in vitro. In addition, this treatment shifted tumor metabolism to a suppressed bioenergetic state in vivo. SLC-0111 also inhibited the enrichment of BTICs after temozolomide treatment determined via CD133 expression and neurosphere formation capacity. GBM xenografts treated with SLC-0111 in combination with temozolomide regressed significantly, and this effect was greater than that of temozolomide or SLC-0111 alone. We determined that SLC-0111 improves the efficacy of temozolomide to extend survival of GBM-bearing mice and should be explored as a treatment strategy in combination with current standard of care.

The mesenchymal phenotype in glioblastoma (GBM) and other cancers drives aggressiveness and treatment resistance, leading to therapeutic failure and recurrence of disease. Currently, there is no successful treatment option available against the mesenchymal phenotype.We classified patient-derived GBM stem cell lines into 3 subtypes: proneural, mesenchymal, and other/classical. Each subtype's response to the inhibition of diacylglycerol kinase alpha (DGKα) was compared both in vitro and in vivo. RhoA activation, liposome binding, immunoblot, and kinase assays were utilized to elucidate the novel link between DGKα and geranylgeranyltransferase I (GGTase I).Here we show that inhibition of DGKα with a small-molecule inhibitor, ritanserin, or RNA interference preferentially targets the mesenchymal subtype of GBM. We show that the mesenchymal phenotype creates the sensitivity to DGKα inhibition; shifting GBM cells from the proneural to the mesenchymal subtype increases ritanserin activity, with similar effects in epithelial-mesenchymal transition models of lung and pancreatic carcinoma. This enhanced sensitivity of mesenchymal cancer cells to ritanserin is through inhibition of GGTase I and downstream mediators previously associated with the mesenchymal cancer phenotype, including RhoA and nuclear factor-kappaB. DGKα inhibition is synergistic with both radiation and imatinib, a drug preferentially affecting proneural GBM.Our findings demonstrate that a DGKα-GGTase I pathway can be targeted to combat the treatment-resistant mesenchymal cancer phenotype. Combining therapies with greater activity against each GBM subtype may represent a viable therapeutic option against GBM.

SUMMARY Unresectable glioblastoma (GBM) cells in the invading tumor edge can act as seeds for recurrence. The molecular and phenotypic properties of these cells remain elusive. Here, we report that the invading edge and tumor core have two distinct types of glioma stem-like cells (GSCs) that resemble proneural (PN) and mesenchymal (MES) subtypes, respectively. Upon exposure to ionizing radiation (IR), GSCs, initially enriched for a CD133+ PN signature, transition to a CD109+ MES subtype in a C/EBP-β-dependent manner. Our gene expression analysis of paired cohorts of patients with primary and recurrent GBMs identified a CD133-to-CD109 shift in tumors with an MES recurrence. Patient-derived CD133−/CD109+ cells are highly enriched with clonogenic, tumor-initiating, and radiation-resistant properties, and silencing CD109 significantly inhibits these phenotypes. We also report a conserved regulation of YAP/TAZ pathways by CD109 that could be a therapeutic target in GBM., Graphical Abstract, In Brief Minata et al., in response to the proinflammatory environment induced by radiation, find that the tumor cells at the invasive edge acquire the expression of the CD109 protein concomitantly losing CD133. CD109 drives oncogenic signaling through the YAP/TAZ pathway, confers radioresistance to the cells, and represents a new potential therapeutic target for glioblastoma.

Expression of c-Myb is required for normal hematopoiesis and for proliferation of myeloid leukemia blasts and a subset of T-cell leukemia, but its role in B-cell leukemogenesis is unknown. We tested the role of c-Myb in p190(BCR/ABL)-dependent B-cell leukemia in mice transplanted with p190(BCR/ABL)-transduced marrow cells with a c-Myb allele (Myb(f/d)) and in double transgenic p190(BCR/ABL)/Myb(w/d) mice. In both models, loss of a c-Myb allele caused a less aggressive B-cell leukemia. In p190(BCR/ABL)-expressing human B-cell leukemia lines, knockdown of c-Myb expression suppressed proliferation and colony formation. Compared with c-Myb(w/f) cells, expression of Bmi1, a regulator of stem cell proliferation and maintenance, was decreased in pre-B cells from Myb(w/d) p190(BCR/ABL) transgenic mice. Ectopic expression of a mutant c-Myb or Bmi1 enhanced the proliferation and colony formation of Myb(w/d) p190(BCR/ABL) B-cells; by contrast, Bmi1 downregulation inhibited colony formation of p190(BCR/ABL)-expressing murine B cells and human B-cell leukemia lines. Moreover, c-Myb interacted with a segment of the human Bmi1 promoter and enhanced its activity. In blasts from 19 Ph(1) adult acute lymphoblastic leukemia patients, levels of c-Myb and Bmi1 showed a positive correlation. Together, these findings support the existence of a c-Myb-Bmi1 transcription-regulatory pathway required for p190(BCR/ABL) leukemogenesis.

Transcriptome profiling of glioma stem-like cells (GSCs) show differences in biological properties depending on their expression signature. Mesenchymal (MES) GSCs are generally associated with increased treatment resistance, thus it is important to identify essential molecules that promote their survival. Here, we discover the preferentially higher expression of the endoplasmic reticulum (ER) chaperone protein glucose-regulated protein 78 (GRP78) in the MES subtype of glioblastomas (GBMs) and derivative GSCs. MES GSCs showed lesser activated basal unfolded protein response (UPR) compared to its proneural (PN) counterparts. GRP78 knockdown in GSCs increased the basal level of UPR and enhanced tunicamycin induced apoptosis. GSCs expressing high levels of GRP78 concomitantly possess higher content of protein and lipid droplets (LDs). As shown by metabolomics studies we have performed in MES GSCs, silencing GRP78 stimulated biosynthesis of lipids that we hypothesize might protect MES GSCs from ER stress induced reactivation oxygen species (ROS) accumulation and apoptosis. In fact, concomitant treatment with simvastatin, a cholesterol inhibitor, induced apoptosis in cells with silenced GRP78 in vitro and significantly improved survival in vivo. Our study shows that higher GRP78 expression and lipid biosynthesis in MES GSC might act as protective mechanisms from ER and oxidative stress, which can be exploited for therapeutic targeting of the MES subtype of GSCs.

IDH-mutant (IDH-MUT) glioma patients show highly improved prognosis compared to those with wild-type IDH. However, IDH mutations are rare in the more aggressive grade IV glioblastomas and introducing IDH mutations in mouse neural cells does not induce gliomagenesis. This is a paradox, given that IDH mutation is considered an early oncogenic event and the mutated IDH produces the oncometabolite 2-hydroxyglutarate. Here we identify a previously unknown mechanism that explains this paradox. We found that IDH-MUT patient tumors expressing a molecular signature enriched in genes associated with neuronal differentiation survive the longest (~11 years) and these same tumors have significantly lower expression of the Mesenchymal master regulator TAZ, which is epigenetically silenced. Concordantly, IDH-MUT/TAZ-low glioma stem-like cells (GSCs) show remarkable neuronal differentiation induced by retinoic acid, whereas IDH-WT/TAZ-high GSCs are resistant to this process implying that IDH status and TAZ levels dictate propensity to neurogenesis. TAZ overexpression in IDH-MUT GSCs caused inhibition of retinoic acid neuronal differentiation and conversely CRISPR/Cas9-mediated knockout of TAZ in IDH-WT GSCs promotes this process as judged by alteration in genes regulating neurogenesis as well as cellular morphology resembling neurons (dendritic branching etc.). Preliminary data on the mechanism of TAZ inhibition, indicate that TAZ might directly act as inhibitor of gene expression via formation of a complex with HDAC-1 and -2. Verteporfin, a FDA approved inhibitor of TAZ caused reduction of tumor volume and improved survival in xenograft bearing mice. We are currently evaluating brain penetrating HDAC inhibitors for their ability to induce neuronal differentiation and tumor inhibition in intracranial models of GBM. Taken together, our studies uncover a role for TAZ as a barrier for terminal neuronal differentiation and that differentiation therapy TAZ or HDAC inhibitors is possible for IDH-WT gliomas if combined with retinoids.

IDH-mutant (IDH-MUT) glioma patients show highly improved prognosis compared to those with wild-type IDH. However, IDH mutations are rare in the more aggressive grade IV glioblastomas and introducing IDH mutations in mouse neural cells does not induce gliomagenesis. This is a paradox, given that IDH mutation is considered an early oncogenic event and the mutated IDH produces the oncometabolite 2-hydroxyglutarate. Here we identify a previously unknown mechanism that explains this paradox. We found that IDH-MUT patient tumors expressing a molecular signature enriched in genes associated with neuronal differentiation survive the longest (~11 years) and these same tumors have significantly lower expression of the Mesenchymal master regulator TAZ, which is epigenetically silenced. Concordantly, IDH-MUT/TAZ-low glioma stem-like cells (GSCs) show remarkable neuronal differentiation induced by retinoic acid, whereas IDH-WT/TAZ-high GSCs are resistant to this process implying that IDH status and TAZ levels dictate propensity to neurogenesis. TAZ overexpression in IDH-MUT GSCs caused inhibition of retinoic acid neuronal differentiation and conversely CRISPR/Cas9-mediated knockout of TAZ in IDH-WT GSCs promotes this process as judged by alteration in genes regulating neurogenesis as well as cellular morphology resembling neurons (dendritic branching etc.). Furthermore, injection of TAZ in the dorsal ectoderm of Xenopus embryos caused a defect in neural development affirming the role of TAZ as a negative regulator of neurogenesis. We are currently examining the mechanisms by which TAZ blocks neurogenesis and the in vivo consequences of concomitant TAZ knockout with retinoic acid treatment in intracranial models. Taken together, our studies uncover a role for TAZ as a barrier for terminal neuronal differentiation and that differentiation therapy with retinoic acid is possible for IDH-WT gliomas if combined with a TAZ inhibitor.

Some cancers including Glioblastoma (GBM) have hierarchical cellular architecture with cancer stem cells (CSCs) as a minor tumorigenic subpopulation. While CSCs have been touted to contribute to initiate recurrence, post-surgical therapy-induced phenotypic changes in CSCs remain elusive. Here we report that treatment of GBM cells with ionizing radiation (IR) promotes a wound-healing mesenchymal signature, accompanied by conversion of naïve CSCs expressing CD133 to post-treatment more radioresistant CSCs expressing the GPI-anchored, TGFb-inhibitory protein CD109 in an ATM, NF-kB, CEBP-b dependent manner. In post-IR GBM, these therapy-escaped CD133-/CD109+ GBM cells, but not the rest of the tumor, uniquely retain clonogenic, tumorigenic, and multi-potent capacities both in vitro and in vivo. CD109 silencing unexpectedly eliminates cancer stemness via controlling YAP/TAZ as the downstream target and this CD109-YAP/TAZ signaling axis is evolutionally conserved between drosophila and human brain tumors. Clinically, both CD133 and CD109 are independently associated with poorer prognosis of GBM patients, while the poorest prognosis group of GBM expressed both CD133 and CD109 at higher rates. These data indicate a possibility of the post-IR CSC plasticity and subsequent heterogeneity, and the activation of the CD109-YAP/TAZ signaling axis as previously unidentified therapeutic targets for post-IR recurrent GBM. Citation Format: Mutsuko Minata, Alessandra Audia, Junfeng Shi, Songjian Lu, Marat Pavlyukov, Yancey Gillespie Gillespie, Krishna Bhat, Ichiro Nakano. Plasticity in heterogenous cancer stem cells promotes glioblastoma radioresistance [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 937. doi:10.1158/1538-7445.AM2017-937

Expression of the transcription repressor Gfi-1 is required for the maintenance of murine hematopoietic stem cells. In human cells, ectopic expression of Gfi-1 inhibits and RNA interference-mediated Gfi-1 downregulation enhances proliferation and colony formation of p210BCR/ABL expressing cells. To investigate the molecular mechanisms that may explain the effects of perturbing Gfi-1 expression in human cells, Gfi-1-regulated genes were identified by microarray analysis in K562 cells expressing the tamoxifen-regulated Gfi-1-ER protein. STAT 5B and Mcl-1, two genes important for the proliferation and survival of hematopoietic stem cells, were identified as direct and functionally relevant Gfi-1 targets in p210BCR/ABL-transformed cells because: (i) their expression and promoter activity was repressed by Gfi-1 and (ii) when constitutively expressed blocked the proliferation and colony formation inhibitory effects of Gfi-1. Consistent with these findings, genetic or pharmacological inhibition of STAT 5 and/or Mcl-1 markedly suppressed proliferation and colony formation of K562 and CD34+ chronic myelogenous leukemia (CML) cells. Together, these studies suggest that the Gfi-1STAT 5B/Mcl-1 regulatory pathway identified here can be modulated to suppress the proliferation and survival of p210BCR/ABL-transformed cells including CD34+ CML cells.

MicroRNA145 (miR145), a tumor suppressor miR, has been reported to inhibit growth of human cancer cells, to induce differentiation and to cause apoptosis, all conditions that result in growth arrest. In order to clarify the functional effects of miR145, we have investigated its expression in diverse conditions and different cell lines. Our results show that miR145 levels definitely increase in differentiating cells and also in growth-arrested cells, even in the absence of differentiation. Increased expression during differentiation sometimes occurs as a late event, suggesting that miR145 could be required either early or late during the differentiation process.

Abstract 1664 Imatinib (IM), a tyrosine kinase inhibitor (TKI), represents the most successful targeted therapy in human cancer. However, this success has been tempered by problems of disease persistence and resistance. We have shown that disease persistence arises from a population of CML stem cells that survive despite complete inhibition of Bcr-Abl by TKI and that in CML stem cells autophagy is induced in response to TKI. Of critical importance, IM-induced autophagy provides a survival mechanism as autophagy inhibition using chloroquine combined with TKIs resulted in almost complete elimination of CML stem cells (Bellodi JCI 2009; Helgason Blood 2011). This pre-clinical work has now progressed to CHOICES clinical trial in which CML patients with residual disease on IM are randomised to continue IM or to have hydroxychloroquine (HCQ) added for 12 months (NCT01227135). In addition to disease persistence, TKI resistance represents a major clinical challenge and can be caused by mutation in Bcr-Abl affecting drug binding (Bcr-Abl dependent) or by activation of alternative signalling pathways (Bcr-Abl independent). As none of the currently licensed TKIs inhibit Bcr-Abl carrying the T315I mutation we aimed to use ponatinib, a 3rd generation TKI, to tackle Bcr-Abl dependent resistance. For cells that have acquired Bcr-Abl independent mechanisms of resistance we aimed to test drugs that target alternative survival pathways and in both models to examine if they induce cell death in TKI-resistant cells, induce protective autophagy and synergise with autophagy inhibition to enhance elimination of CML stem/progenitor cells. We demonstrated that ponatinib was effective in inhibiting proliferation and inducing apoptosis in chronic phase (CP) CML CD34+ cells (n=5) and in CP cells carrying the T315I mutation (n=2) in a dose-dependent manner. In addition, ponatinib, like 1st and 2nd generation TKIs, induced autophagy in CP CD34+ and T315I expressing cells. HCQ-mediated autophagy inhibition enhanced the effect of ponatinib in CML stem/progenitor cells with reduction in Colony Formation Cell (CFC) number increased from 34% with 100nM ponatinib alone to 77% in ponatinib/HCQ treated cells (n=4). This combination had no significant effect on proliferation or CFC formation of normal CD34+ cells (n=3). Most strikingly, ponatinib/HCQ combination resulted in near complete elimination of CML stem cells with less that 1% surviving treatment in Long-Term Culture Initiating Cell (LTC-IC) assay (n=3). The PI3K/Akt/mTOR pathway, downstream of Bcr-Abl, provides an alternative drug target for CML patients with Bcr-Abl independent TKI-resistance. Treatment with clinically relevant concentrations of the dual PI3K/mTOR inhibitor BEZ235 resulted in dose-dependent induction of apoptosis in CP CML CD34+ cells (n=5) and in ponatinib-resistant KCL22 cells (generated by culturing cells in increasing concentrations of ponatinib for a prolonged period), whereas rapamycin, a less potent mTOR inhibitor, exhibited only anti-proliferative effects and failed to induce apoptosis. We also showed that inhibition of mTOR leads to induction of autophagy. Furthermore, autophagy inhibition, by Atg7 knockdown or HCQ treatment, augmented death induced by PI3K/mTOR inhibitors in CML cells, including ponatinib-resistant KCL22 cells. In CP CD34+ cells 100nM BEZ235 induced 12% apoptosis above untreated controls (n=6) and 65% reduction in CFC (n=8), that was increased to 18% and 83%, respectively, when combined with HCQ. When combined with HCQ-mediated autophagy inhibition, BEZ235 also reduced CFC potential of progenitor cells derived from 2 patients who have failed to achieve CCyR following any generation TKI, by 75% compared to 42% following ponatinib treatment. BEZ235 as monotherapy reduced survival of CML stem cells by 59% with the effect further increased in combination with HCQ, resulting in 74% reduction in stem cell number, indicating that autophagy also protects CML stem cells from death induced by PI3K/mTOR inhibition. Taken together these data indicate that autophagy inhibition might not only potentiate treatment for CML patients responsive to TKI treatment by enhancing elimination of CML stem cells, but may represent an improved treatment option for both Bcr-Abl dependent and independent mechanisms of TKI-resistance in CML patients. Acknowledgments: Grants from KKLF (KKL404) and MRC (G0900882, CHOICES, ISCRTN No. 61568166) Disclosures: Nicolini: Novartis, Bristol Myers-Squibb, Pfizer, ARIAD, and Teva: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Druker:MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Scientific Founder; OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. This potential individual and institutional conflict of interest has been reviewed and man, Scientific Founder; OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. This potential individual and institutional conflict of interest has been reviewed and man Other; Novartis: OHSU receives clinical trial funding, OHSU receives clinical trial funding Other; Bristol-Myers Squibb: OHSU receives clinical trial funding, OHSU receives clinical trial funding Other; ARIAD: OHSU receives clinical trial funding. Dr. Druker is currently principal investigator or co-investigator on Novartis, Bristol-Myers Squibb, and ARIAD clinical trials. His institution has contracts with these companies to pay for patient costs, nurse and da Other.