Targeting NF-KB Activation in Novel Intracranial Models of CNS Lymphoma

Among the clinical subtypes of diffuse large B-cell lymphoma, central nervous system (CNS) lymphomas, both primary (PCNSL) and secondary (SCNSL), are generally associated with the worst prognosis. Moreover, PCNSL typically exhibits multifocal dissemination, prompting its description as a whole brain disease. An accumulation of genomic data suggests that aberrant NF-kB pro-survival signaling may be a critical factor in the pathogenesis of CNS lymphoma. To date, however, there has been an absence of mechanistic studies to interrogate the mediators and significance of NF-kB activation in CNS lymphoma invasion. Recently our lab derived the first panel of xenografts yet established from PCNSL patients (n=7), and defined their invasive properties in murine models. These novel model systems provide a unique opportunity to study key regulatory mechanisms as well as novel targeted therapies of CNS lymphoma invasion and growth within the brain.

We determined that the intracranial growth patterns of the xenografts can be classified based upon reproducible patterns of invasiveness. Two xenografts derived from SCNSL failed to infiltrate brain parenchyma or to migrate to the contralateral hemisphere; instead these tumors exhibited solely a meningeal dissemination pattern, largely growing in the ventricles. By contrast, five of the xenografts exhibited diffuse brain invasion into bilateral hemispheres. In addition, we noted that CNS lymphoma xenografts with an invasive phenotype can be sub-divided phenotypically into moderately vs. highly invasive tumors. After standard injection of 200,000 tumor cells/brain, the highly invasive lymphomas were reproducibly lethal to mice within 3 weeks post-implantation and yielded ~ 1X10⁶ lymphoma cells/brain at necropsy. Under identical conditions, the moderately-invasive lymphoma xenografts were lethal to mice within 1-3 months and yielded between 3-5 X10⁶lymphoma cells/brain. Notably each of the highly invasive CNS lymphoma xenografts were derived from CNS lymphomas that contained the activating L265P mutation of MYD88 (activator of NF-kB via IRAK1/4) and/or Y196H mutation of CD79B (activator of NF-kB), whereas the non-invasive xenografts contained wild type MYD88 and CD79B.

We analyzed RNA-seq expression data of these xenograft samples to evaluate relationship of individual genes and signaling pathways with the distinct subclasses of CNS lymphomas in terms of invasiveness. Upregulated signatures in the pro-invasive phenotype are significantly enriched with NF-kB target genes (http://lymphochip.nih.gov/signaturedb/) (FDR<3e-4). For example, as expected, the most invasive xenografts derived from CNS lymphoma specimens expressed high RelA (p65) and NFKB1, core components of the NF-kB multimeric complex. The invasive CNS lymphoma xenografts also exhibited upregulation of MYD88, SYK and PIM1/2, inducers of NF-kB activation. Conversely, the non-invasive xenografts expressed a signature of predominantly non-NF-kB-induced genes including SPRY1/2 (negative regulators of receptor tyrosine kinases) and TIMP2 (negative regulator of invasion). Gene set enrichment analysis confirmed a similar pattern of transcripts expressed in highly invasive PCNSL isolated from diagnostic specimens in patients (N=23; FDR <0.017).

Given that the L265P mutation of MYD88 represents a candidate key driver of NF-kB activation in CNS lymphoma, we evaluated the preclinical efficacy of AZ1495, a novel and potent inhibitor of IRAKs. We demonstrated CNS penetration of AZ1495 after PO administration to mice with intracranial lymphoma xenografts, yielding a mean brain/plasma partition coefficient of 0.8. AZ1495 potently antagonized NF-kB activation as demonstrated by p-p65 immunoblot analysis of tumor lysates isolated from murine CNS lymphoma xenografts, within 3 hrs of administration. AZ1495 monotherapy resulted in > 2-fold delay in both intracranial plus spinal progression in a dissemination model of CNS lymphoma using L265P mutant OCI-LY10 cells engineered for bioluminescence (p<0.01). Finally, AZ1495 treatment resulted in a >2-fold increase in median OS in a patient-derived model of L265P mutant PCNSL. These results support the preclinical utility of these models to dissect impact of NF-kB inhibition on CNS lymphoma progression as well as the clinical potential of IRAK antagonism as a means to target NF-kB activation in L265P mutant CNS lymphomas.