Nicholas Szerlip, MD Department of Neurosurgery Wayne State University Karmanos Cancer Institute

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1 Nicholas Szerlip, MD Department of Neurosurgery Wayne State University Karmanos Cancer Institute

2 Many different names (Heregulin, Aria) Found when investigating ligands that activated HER2 (it actually does not bind to HER2) Heregulin- purified from conditioned breast tumor media (promoted cell growth) Plays a role in normal growth and development

3 Ligand (growth factor) specific for the Epidermal Growth Factor (EGF) family Many different isoforms Each has different tissue specificity

4 NRG activate HER receptors Four HER receptors HER1-4 NRG1 has affinity for binding HER4>Her3 HER2 has no known ligand that binds to its receptor When NRG1 binds to HER3 or HER4 conformation changes and an extracelluar dimerization arm is formed. Receptor must heterodimerize to activate/phosphorlyate

5 It will heterodimerize with another activated HER receptor whose arm is exposed or with any HER2 receptor. Therfore statisticaly HER2 receptor is the preferred dimerization partner in this pathway.

6 Synthesized by cells as membrane bound growth factors with the EGF domain exposed They will either remain on membrane or be solubolized to interact with other cells and/or tissues This occurs via cell surface proteases

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8 Most important functions of NRG are in development Cardiac, Central Nervous System (Shwann cells, neurons, myelin formation lots of others things) A lot of these functions have to do with cell migration It has been implicated in many disease processes Schizophrenia, MS, TBI, Angiogenesis, Wound healing, Tumor formation. Shown to be important in many cancers Breast Cancer 20-90% Prostate 50-90% Medulloblastoma 90%

9 Neuregulin has been shown to have mitogenic properties on tumor cells via autocrine and paracrine signaling Has been shown to increase tumor cell migration and decrease apoptosis Non-tumor models have shown neuregulin has a direct effect on microglia growth and proliferation

10 Why might this be an important player in Primary Brain tumors

11 A primary brain tumor originating from support cells of the brain Account for 80% of primary brain tumors Occurs in patients age Result in more years of life lost than any other tumor Associated with poor survival: life expectancy of months after diagnosis

12 More molecular information collected about Glioblastoma than any other tumor No information has translated into significant clinical benefit Current treatment paradigm remains uniform Surgical debulking followed by radiation and temozolamide

13 Infiltrating tumor margins makes complete resection Microscopic tumor infiltration Visible tumor impossible

14 GBM Tumor Touch Prep EGFR PDGFRA

15 Michael Berens (T-Gen) Showed gene differences in core of tumor vs. infiltrating brain Overall we have a poor understanding of this heterogeneity and how different oncogenic pathways work in this paradigm

16 In addition to the interaction of tumor cells with different molecular signatures is the interaction of the tumor cells with the immune cells (microglia,macrophages) Over the last decade a substantial role for microglia/macrophages subtypes has been found in tumor progression but not fully ellucidated

17 Gliomas show lots of infiltration of microglia/macrophages ( up to 30% of tumor mass) There is a correlation between grade and abundance of these cells These cells release a host of cytokines and proteases that play a role in tumor progression and depletion of these cells effects tumor invasiveness

18 Has been shown to increase migratory ability of both tumor cells and microglia cells Hypothesis: NRG is present in and plays a role in the migratory phenotype of GBM Elucidation of this system will lead to a better understanding of the interaction between glioma cells and microglia/macrophages Targeting NRG is a means of targeting cancer cells and their support cells in a clinically meaningful way

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20 Literature is divided Some say it is not present in gliomas, some say it is Most studies looked at only cell lines (some had it some did not) and one or two biopsy samples

21 Total Her2 Total Her3 Total Her4 SC-348 Tubulin

22 12-49 tumor core Gad + SC348 Green, CD68 RED

23 12-49 GBM tumor core Gad + Green PHER2, RED CD68

24 Total Her2 P-Her2 Tubulin

25 M1 Phenotype (CD80/196+) TAM Phenotype (CD 68+ not M1 or M2) HER2 (-) M2 Phenotype (CD206/163+)

26 13-05

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28 Total Her2 P-Her2 Tubulin Percent Change U251+NRG NRG 12-38G+NRG NRG

29 We have shown that for the NRG is present at variable levels in GBMs It is produced by Cancer cells and Microglia/macrophages in different amounts depending on as of yet unkown variables The tumor cells may self activate their HER receptors or get them activated by another GBM cell or microglia

30 The overall objective of this research program is 1) Define the NRG1 signaling pathway in-vivo and determine how radiographic differences correlate to differences in this pathway and microglia phenotype 2) Quantify the effects of NRG1 on the interaction between glioma cells and microglia subtypes (M1, M2) 3) Test a novel inhibitor of this pathway for stratification of future targeted therapeutics

31 Comments

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33 We hypothesize that NRG1 plays an autocrine/paracrine role in glioma growth and migration by effecting the glioma cell population as well as the microglia population which in turn has a proliferative/migratory effect on glioma cells and that this system will vary topographically within a tumor corresponding to phenotypic differences of the cells (glioma, microglia).

34 Aim 1. Characterize the neuregulin signalling pathway in gliomas of different types/grades topographically depending on the radiographic imaging characteristics. Examine the penetrance of NRG and its receptor activation in a set of gliomas Correlate this to transcriptional classification scheme

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36 Quantify NRG and Her2 expression in different regions Immunofloresence Western blot FACS Examine this expression by function of cell type (glioma cell Vs. Microglia) as well as the regional microglia/macrophage phenotype differences and their Her2 expression FACS

37 Determine the functional effects of neuregulin pathway on growth proliferation, and migration of glioma cells and microglia/macrophage phenotypes and examine the differences between in vivo low expressers vs high expressers.

38 Using known cell line (U251) and patient derived cell lines explored in Aim1 (high and low expressers of NRG/Her2) Examine growth/proliferation assays (MTT) migration (Boyden chamber) Examine in isolation of microglia with and without neuregulin Block neuregulin activation with a novel inhibitor developed in our lab as well as sirna to stop neuregulin production

39 We will examine the effects of neuregulin on microglia/macrophage growth/ proliferation and migration in a similar manner Isolate peripheral blood monocytes from normal subjects Grow and skew to either M1, M2 or TAM Determine the amount of endogenous neuregulin production in these different states and examine the effects of blocking neuregulin on growth, proliferation and migration Obtain microglia/macrophages from tumor samples by FACS and grow and skew as before

40 We will test the validity of using a xenograft model and test this novel drug that is known to block neuregulin activation of Her-2 in such an animal model of brain cancer

41 Nude rat xenograft model with different tumor types high/low Her2 expressers. Using an osmotic pump and cannula to convect drug into injection site at time of initial injection or when tumor is fully formed. Drug will be administered via pump by osmotic delivery in a steady state for thirty days

42 We will assess tumor growth rate and survival. After thirty days animal will be sacrificed and tumors will be examined for expression level of her2 and microglia/macrophages will be quantified and characterized

43 These results will give an understanding of role of NRG signalling pathway Microglia/macrophage interaction with glioma cells Depending on phenotype of glioma cell/microglia Allow us to assess this pathway as a main therapy or adjuvant therapy. Give us insight to develop other targets for treatment both targeted molecular treatments pathway specific as well as how this interaction effects the immune cells for immune therapy

44 Elucidate this pathway organization in different glioma subtypes and the effect of blocking this pathway in glioma subtypes

45 The information will give us insight into the role of the different phenotypes of microglia/macrophages that will allow us to continue to explore how tumor cells effect their microenvironment and what targets are present to take advantage of in treatment

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47 Show variation of Her2 in GBMs tissue by western High and low expressers Have cell culture correlates Show variation in flow data with different HER2 activation by Microglia/Macrophage Phenotype in different GBMs Show neuregulin release in different GBM cell lines Show inhibition of Neuregulin with Gly-B4 leads to inhibition of HER2 phosphorylation

48 Neuregulin Promotes Breast Cancer Growth

49 Identify and target different subpopulations Either concurrently or sequentially Difficult due to toxicity Identify targets that are molecularly altered in all cells Difficult because targets are likely found in normal cells Identify targets that are upregulated by interaction of cell and tumor environment I.E. a selectively inducible system Less reliant upon molecular uniformity of cancer genome more reliant of the metabolic nature of a cancer cell Less dangerous to normal cells

50 This is a simplified classification scheme that puts microglia/macrophages into two phenotypes M1-pro-inflmmatory(induced by IFN-gamma and LPS) Neuro-toxic M2 anti-inflammatory (induced by IL-4 or IL-13) Promote a regenerative growth response

51 These cells are activated by morphology but fail to express MHC II markers they lack surface markers associated with M1 phenotype and do not respond like M1 as far as cytokine secretion goes.

52 Lack of durable response may be due to a combination of molecular heterogeneity between tumors that have been grouped into a study as well as molecular heterogeneity within a tumor