Novel Targeted Combination Therapies for Peripheral T-Cell Lymphoma
Authors: Mahadevan Daruka , Morales Carla , Huber Bryan D. , Ul Haq Mansoor , Spier Catherine M. , Cooke Laurence S. , Qi Wenqing 
Published: October 2019

Abstract
Introduction : New and promising evidence indicates that Aurora A and B kinases are important drivers of peripheral T-cell non-Hodgkin lymphoma (PTCL), a rare and heterogeneous disease accounting for ~15% of non-Hodgkin lymphomas (NHLs), without a clear standard of care. Auroras are a highly conserved family of oncogenic serine/threonine protein kinases that play key regulatory roles during mitosis. They are over-expressed and dysregulated in malignancy which make them very attractive therapeutic targets. We investigated the molecular, cellular, and cytokine drivers of PTCL in patients (S1108) treated with an aurora A inhibitor, alisertib (MLN8237). It is hypothesized that patient and pre-clinical models will guide the design of better strategies to target auroras in PTCL.
Methods : Immunohistochemistry (IHC) on 22 PTCL patient samples (S1108) for level of expression of aurora A, aurora B, Myc, Notch-1, Bcl-2 and PI3Kd was performed. Nuclear positivity was determined for aurora A, B and Myc using Aperio’s Nuclear v9 Quantification algorithm. Expression of Bcl-2, Notch-1 and PI3Kd was quantified with Aperio’s Positive Pixel Count algorithm. The Raybiotech G2000 human cytokine array of 174 unique cytokines was analyzed using pre-/post-alisertib (day 8) therapy from 12 PTCL patient samples. MTS cellular cytotoxicity assays were conducted with single agent alisertib, PI3K inhibitor (MLN1117), mTor inhibitor (MLN0128) and vincristine (VCR) in TIB-48, CRL-2396 and Jurkat T-NHL cell lines. Combination studies were conducted with alisertib + MLN1117 or MLN0128 or VCR or triple combinations to ascertain IC50s and synergism (Chou-Talalay). A mouse PTCL xenograft model is underway to evaluate the anti-tumor activity of alisertib, MLN1117, and alistertib + MLN1117. Mechanistic studies were conducted for target and pathway inhibition.
Results : As previously shown in patients from S0350, S1108 confirms aurora B is over-expressed in most patients (nuclear) versus aurora A (cytoplasmic). PI3Kd is over-expressed on the surface and/or the cytoplasm mainly within the tumor microenvironment. Myc expression is nuclear within a host-response type infiltrate in the tumor. Bcl-2 expression is confined to small T-cells, a host-response marker. Notch-1 expression was only observed in 2 cases within large tumor cells. Although level of expression of the 6 IHC markers did not correlate with alisertib activity, aurora B and PI3Kd are highly over-expressed in PTCL. Cytokine profiling revealed that pre-/post-alisertib is unique to each patient and are correlated to therapeutic response. Of note 4 of 12 patients had an elevated soluble TNF-RII, a poor prognostic marker in PTCL. IC50s for alisertib in CRL-2396, TIB-48 and Jurkat cells: 44-64 nM, MLN1117 9-14 μM, and MLN0128 36-130 nM. Combination indices (CI) indicate strong synergism for alisertib +MLN1117, MLN0128+MLN1117 and alisertib+MLN1117+VCR. These combinations lead to target inhibition and apoptosis. The PTCL xenograft mouse safety and response data will be available at the meeting.
Conclusions : Aurora and PI3Kd inhibition + VCR holds promise as a combination targeted approach for patients with PTCL. IHC to stratify and cytokines to follow therapy are biomarkers to be considered in prospective trials in the treatment of PTCL patients.

Disclosures
No relevant conflicts of interest to declare.