Ex Vivo Multiplexed Signal Pathway Inhibitor Treatment Reveals Differential Sensitivity of Myeloma and Non-Myeloma Bone Marrow Cell Populations.

Abstract 2860

Poster Board II-836

Multiple Myeloma (MM) is a heterogeneous disease, and despite significant advances in therapy it remains incurable. A major challenge facing clinicians is to determine which new agent or which combinations of agents will prove efficacious for individual patients.

Interaction of MM cells with bone marrow microenvironment cells has a pathogenetic role in the disease and confers tumor cell resistance to conventional therapies. The new generation of therapies is designed to target signal transduction pathways, particularly tyrosine kinase signaling. The phosphorylation, or activation state, of kinase-driven signal networks contains important information concerning disease pathogenesis and the ongoing state of kinase-associated therapeutic targets. Reverse-phase protein microarray (RPMA) is a reproducible, high-throughput system for protein signal pathway profiling. Phosphorylation state of kinase-associated therapeutic targets provides direct information regarding the target and off-target effects of treatment.

We describe herein an ex vivo study of signal pathway inhibitor treatments of fresh bone marrow aspirate samples from patients undergoing standard of care hematological work up for multiple myeloma at any stage or treatment course. Bone marrow aspirates (n=37 to date), not required for diagnosis, were immediately subdivided and treated ex vivo with a panel of molecular targeted inhibitors and combinations, and/or exogenous ligands (SCF, IGF-1), and/or cytokines (IL-6), that target a wide range of cellular pathways (e.g. proteosome, angiogenesis related, protein degradation, cell proliferation/survival, insulin response, and protein translation). Up to 48 different treatment conditions can be studied for each patient sample from a bone marrow aspirate volume of 5.0mL. The objectives were a) measure the signal pathway perturbations caused by the inhibitor/ligand treatment in individual bone marrow aspirate samples, b) compare the relative sensitivity of tumor and non tumor bone marrow cells treated in admixture under identical conditions to identify predictive/prognostic protein-based biomarkers. We specifically measured the post-translational modifications (i.e. phosphorylation) of the drug target substrates to provide direct information regarding the target and off-target effects of treatment.

After incubation for 4 hours the bone marrow aspirate samples were placed in a preservative that suppresses fluctuations in kinase pathway proteins. MM CD138 positive cells were separated from the non-CD138+ bone marrow microenvironment cells via immunomagnetic sorting. RPMA was used to quantitatively map 75 cell signaling pathway endpoints in CD138+ and non-CD138+ cell populations that were treated simultaneously prior to cell sorting.

To date the accrual has included untreated patients with MGUS, smoldering myeloma, treatment naïve, or advanced stage, pre-treated myeloma. Individual patients differed widely in response to ex vivo treatment and combination of drug treatments. Dexamethasone showed differential effects on the myeloma cells compared to non-myeloma cells from the same patient. For all the untreated patients studied to date, dexamethasone significantly suppressed the phosphoAKT or NF-KB endpoints, while increasing the pro-apoptotic signaling proteins such as cleaved caspases. In contrast, all the patients heavily pre-treated with dexamethasone in combination with other agents did not show any differential effects of the dexamethasone treatment on the CD138+ cells compared to the non-CD138+ cells in the bone marrow microenvironment.

Many molecular targeted inhibitors induced an up-regulation of the target pathway even in combination therapies such as Rapamycin plus an IGF-1R inhibitor. Sorafinib, a molecular targeted tyrosine kinase inhibitor which blocks the MAPK (Raf/Mek/ERK) pathway, induced a compensatory up-regulation of ERK T202/Y204 in all patients except one who was in remission after treatment.

Multiplexed phosphoprotein cell signaling analysis before or after treatment with ex vivo inhibitors may predict patient-specific therapeutic response and/or off target effects. Selected agents that show efficacy in ex vivo studies may be considered for further in vivo clinical studies in which signal profiling of the bone marrow cellular populations could be performed before and after molecular targeted therapy.