Melanoma Cell Biology

Project 1: Examine genetic effectors that modulate response of BRAF-mutant melanoma to combination BRAF and MEK inhibitors

Introduction

Until recently, patients with metastatic melanoma were treated with single agent chemotherapy drugs that produce response rates of less than 10%, with no improvement in overall survival. New drugs targeting the mitogen activated protein kinase (MAPK) pathway have now shown significant activity. These drugs were developed because MAPK signalling is constitutively activated in most melanomas through oncogenic mutations in either NRAS (10-30% cutaneous melanomas) or its downstream kinase effector BRAF (~50%).

Despite the dramatic clinical activity of combining BRAF and MEK inhibitors, only 41% of patients on combination therapy were progression-free at 1 year.

Hypothesis:

  1. The MAPK cascade is the central proliferation pathway in melanomagenesis and its re-activation occurs in most melanoma tumours with acquired resistance to BRAF inhibitor monotherapy. We propose that:
  2. Melanoma tumours circumvent BRAF and MEK inhibitor combination therapy by reprogramming and reactivating the MAPK network, and
    Pre-existing cancer mutations diminish cancer cell dependence on oncogenic BRAF signalling.

Aims:

  1. Define the genetic and signalling changes associated with resistance in vivo, by:
    1. Examining genome sequencing data of melanomas progressing on combination BRAF and MEK inhibitors
    2. Investigating changes in the signalling pathway activity of melanomas biopsied early during therapy
  2. Examine the utility of circulating DNA as a predictor of patient response and resistance

Research Plan:

Next generation exome sequencing of patient-matched PRE-therapy and Progressing melanoma tissues, will be performed on >20 tumour pairs. A well-established filtering strategy will be used to identify mechanisms of resistance and modulators of response. Early responses to MAPK inhibition (comparing PRE-therapy and EDT (early during therapy) melanoma tissue) will also be examined as a means of defining early changes in signalling activity.

A series of functional assays will be used to validate candidate gene variants. As an example of our functional validation work, we are currently analyzing the role of the MEK1 and MEK2 mutants. We have generated epitope tagged versions of the wild type and mutant MEK genes and they will be introduced into two BRAF-mutant, BRAF inhibitor-sensitive human melanoma cell lines. We are using the tetracycline-inducible lentiviral expression system to regulate transgene expression and allow for comparison of "physiologic" and high levels of ectopic MEK accumulation. The impact of ectopic MEK expression on cell sensitivity to inhibitors of BRAF, MEK and/or ERK will be investigated using clonogenic assays, MTT viability assays and annexin V apoptotic assays. Changes in MAPK signalling will be measured by immunoblotting key markers of pathway activity, including p-ERK, cyclin D1, DUSP6 and p27Kip1 accumulation.

The response of patients (n=35) to combination BRAF/MEK inhibitors will also be monitored during the course of 12-week therapy using highly sensitive digital droplet PCR.

Enquires:
Professor Helen Rizos

Email: helen.rizos@mq.edu.au

Project 2: Examine the role of the tumour/immune microenvironment on melanoma response to targeted therapy

Introduction

Melanoma is a major public health problem in Australia with rising incidence and mortality, and for patients with metastatic melanoma prognosis remains poor.
The mitogen activated protein kinase (MAPK) pathway is a critical proliferation pathway in melanoma and is frequently activated via mutations in BRAF (present in 50% of melanomas), NRAS (20%) and KIT (20% of mucosal melanoma).  Dual inhibition of the MAPK pathway using combinations of BRAF and MEK inhibitors has recently become the standard of care for treating patients with advanced BRAF-mutant advanced melanoma, and is being tested in clinical trials in the neo-adjuvant and adjuvant settings for earlier stages of melanoma at Melanoma Institute Australia (MIA).  Although 70% of patients respond to combination BRAF and MEK inhibition, most will develop resistance and the median progression free survival is only 9.4 months.  Therefore, further research is urgently required to optimise treatment.
It is well documented that melanoma is an immunogenic tumour, and the presence of T-cell infiltrates within melanoma lesions correlates positively with longer patient survival.  Translational studies have shown that tumour infiltrating lymphocytes (TILs) play an important role in determining response to targeted therapies however the precise mechanisms remain to be elucidated.  

Hypothesis:

Melanoma patient response to targeted therapy is influenced by the tumour microenvironment. We propose that cytokine secretion profiles and the degree and type of immune cell infiltration modulate response to targeted therapies.

Aims:

  1. Examine changes in the immunophenotype of tumour infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs) during treatment with BRAF and MEK inhibitors.
  2. Examine the cytokine profile of melanomas and plasma during the course of treatment with targeted therapy
  3. Correlate immunophenotype and cytokine changes with patient responses to therapy  
  4. Identification of tumour neo-antigens in patients showing durable responses   

Methods:

This project will use data generated from analysis of tumours and paired blood and plasma from 35 melanoma patients enrolled in a phase II study being run at Melanoma Institute Australia.  Whole genome microarray analyses will be used to explore how immune-related tumour expression signatures vary during the course of treatment.  Results will be confirmed using RT-PCR, Western blotting and ELISAs, as required.  Flow cytometry will explore the immunoprofile of melanoma cells, TILs and PBMCs during the course of treatment. Cytokine-profiles from tumours, PBMCs and plasma will also be determined using ELISA assays over the treatment course.  The influence of tumour genotype (determine using whole genome sequencing) on patient response, cytokine secretion and immune infiltration will also be examined. Tumour neo-antigens will be explored in a subset of complete responders using patient-derived TILs and COS7 cells transfected with tandem minigenes encoding tumour mutations.

Enquires:
Professor Helen Rizos

Email: helen.rizos@mq.edu.au

Project 3: Manipulating oncogenic signalling for the treatment of KIT-mutant melanoma

Introduction

The receptor tyrosine kinase KIT is activated by mutation and the wild type KIT gene is also commonly amplified in many types of human cancer. More than 80% of gastrointestinal stromal tumours (GISTs) display KIT mutations, and 20% of melanomas arising on mucosal membrane and acral skin have activating mutations in KIT. Kinase inhibitors that block KIT have revolutionised the treatment of GIST. More than 80% of KIT-mutant GIST patients respond to the KIT inhibitor imatinib and most patients will live for more than 5 years compared to only one year pre-imatinib. KIT-mutant melanoma patients also respond to imatinib (response rate ~30%), but most patients will progress within 4 months.

It is unclear why the response of KIT-mutant melanoma patients to imatinib is so short-lived. Most melanoma and GIST-associated KIT mutations occur in exon 11, and although there is a preponderance of the activating L576P mutation in melanoma this mutation does not predict response; patients with KITL576P melanoma can have complete durable responses or show no clinical response to imatinib3.

Hypothesis:

We propose that most KIT-mutant melanomas are refractory to KIT inhibitors because pre-existing cancer mutations diminish cancer cell dependence on oncogenic KIT signalling. This hypothesis reflects evidence that the probability of resistant cancer subclones existing prior to therapy is proportional to the size of the tumour and the tumour mutation rate. Patients with advanced melanoma have a high tumour burden and melanomas have elevated baseline mutation rates.

Aims:

  1. Discover genetic effectors that diminish KIT-signalling dependence in melanoma
  2. Define additional oncogenic activities in KIT-mutant melanoma
  3. Identify novel treatments for KIT-mutant melanoma

Research Plan:

This project utilises patient specimens, melanoma cell and xenograft models to examine why most melanomas with activating KIT mutations are refractory to current therapies. In order to establish the frequency and nature of genetic changes that limit melanoma cell dependence on KIT activity we will sequence and conduct whole genome microarray analyses of 10-20 KIT-mutant melanomas. We will apply a series of functional assays to validate each novel resistance driver with a particular focus on pathways rather than analyzing every potential gene mutation. The impact of each introduced gene on cell sensitivity to inhibitors of KIT will be investigated using clonogenic assays and in cells grown as spheroids. Once melanoma-associated gene mutations that influence KIT-dependence are identified, we will examine the activity of rational therapies in vitro and in mouse models. We will also perform (via the ACRF Drug Discovery Centre at the Children's Cancer Institute) an unbiased drug screen in KIT-mutant melanoma cells to 3707 FDA-approved chemotherapeutic agents and 210 novel kinase inhibitors in four KIT-mutant human cell lines.  Effective treatments for KIT-mutant melanoma are urgently needed, and this drug screen be rapidly translated into the clinic.

Enquires:
Professor Helen Rizos

Email: helen.rizos@mq.edu.au

Project 4: Evaluate the role of MAPK-independent pathways in regulating melanoma responses to combination BRAF and MEK inhibitors

Introduction

The serine/threonine kinase BRAF is constitutively activated via mutations in 50% of cutaneous melanomas and causes constitutive activation of the mitogen-activated protein kinase (MAPK) cascade. The combined inhibition of mutant BRAF and its downstream target MEK, have produced response rates of 70%, and prolong the progression-free and overall survival of patients with BRAF-mutant melanoma, compared with chemotherapy.
Despite the dramatic clinical activity of combining BRAF and MEK inhibitors, only 41% of patients on combination therapy are progression-free at 1 year. Resistance mechanisms reactivating the MAPK pathway are common but a subset of resistant tumours show evidence of durable MAPK inhibition, and the outcome of patients with these tumours is poor.

Hypothesis:

We propose that MAPK signalling must be re-established for resistance to MAPK inhibitors and activation of alternate, survival networks provides an opportunity for the acquisition of MAPK-reactivating mechanisms. We also suggest that the degree of MAPK activity reflects the network of positive and negative regulatory loops, and thus varies in resistant melanomas.

Aims:

  1. Examine the role of alternate signalling activity on the response of BRAF-mutant melanoma cells to combination BRAF and MEK inhibitors
  2. Develop and analyse melanoma cells resistant to BARF and MEK combination therapy with inhibited MAPK activity
  3. Explore MAPK signalling at the protein level in small subset of 'MAPK-inhibited' resistant tumours, and validate any novel mechanisms defined in Aims 1 and 2.

Research Plan:

This project will explore the activation of key signalling pathways (selectively activated via ectopic expression of mutant AKT, PI3K, APC or JNK etc or downregulation of selected genes such as p16INK4a, Rb1, PTEN etc) in a panel of BRAF-mutant, BRAF inhibitor-sensitive human melanoma cell lines. The impact of pathway activation on the proliferation and survival of cell lines will be explored using clonogenic assays, MTT viability assays and annexin V apoptotic assays. Changes in pathway signalling will be measured by immunoblotting key markers of pathway activity, including p-ERK, cyclin D1, DUSP6 and p27Kip1 accumulation and protein arrays.

In order to model resistance in the absence of MAPK activity, we will expose single cell-derived clonal melanoma sublines to combination BRAF, MEK and ERK inhibitors, and identify clones that develop resistance while MAPK activity is switched off. Resistant clones will be grown for at least 25 passages in the presence or absence of the drug and the stability and degree of resistance will be measured by the MTS colorimetric assay of culture growth. Mechanisms of resistance will be evaluated using a combination of exome and RNA sequencing.

Any MAPK independent genetic effectors of resistance will be validated in a small cohort of progressing melanomas with evidence of MAPK-indpendent resistance.

Enquires:
Professor Helen Rizos

Email: helen.rizos@mq.edu.au