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The complexity of heparin and heparan sulfate saccharides makes their purification, including many isomeric structures, very challenging and is a bottleneck for structure-activity studies. High-resolution separations have been achieved by strong anion exchange (SAX) chromatography on Propac PA1 and cetyltrimethylammonium (CTA)-C18 silica columns; however, these entail subsequent desalting methodologies and consequent sample losses and are incompatible with orthogonal chromatography methodologies and, in particular, mass spectrometry. Here, we present the CTA-SAX purification of heparin oligosaccharides using volatile salt (VS) buffer. In VSCTA-SAX, the use of ammonium bicarbonate buffer for elution improves resolution through both weaker dissociation and conformational coordination of the ammonium across the sulfate groups. Using ion mobility mass spectrometry, we demonstrate that isomeric structures have different structural conformations, which makes chromatographic separation achievable. Resolution of such structures is improved compared to standard SAX methods, and in addition, VSCTA-SAX provides an orthogonal method to isolate saccharides with higher purity. Because ammonium bicarbonate is used, the samples can be evaporated rather than desalted, preventing substantial sample loss and allowing more effective subsequent analysis by electrospray mass spectrometry. We conclude that VSCTA-SAX is a powerful new tool that helps address the difficult challenge of heparin/heparan sulfate saccharide separation and will enhance structure-activity studies.

Background: The Ikaros family zinc finger transcription factor, Aiolos, is upregulated in more than 80% of CLL patients with data suggesting that it may be an important driver of CLL survival and hence a bona fide target for therapeutic intervention. To date, transcription factors such as Aiolos have not been amenable to drug therapy. We and others have shown that the IMiDs™ immunomodulatory drugs such as Lenalidomide (LEN) bind Cereblon (CRBN), a substrate receptor of the cullin ring E3 ubiquitin ligase complex (CRL4CRBN) and promote ubiquitination and subsequent proteosomal degradation of lymphoid transcription factors Ikaros and Aiolos. Such insights have been critical for the design and development of next generation IMiD analogues. CC-122 is a novel non-phthalimide, first-in-class, PPM™ pleiotropic pathway modifier drug with anti-proliferative, anti-angiogenic, and immunomodulatory properties. CC-122 specifically targets CRBN and promotes degradation of Aiolos and Ikaros. We have now investigated the effects of LEN and CC-122 on CLL proliferation, and Aiolos and Ikaros degradation, in a cohort of primary CLL cases using an in vitro co-culture system based on CD154 expressing mouse fibroblasts and exogenous IL-21 that recapitulates the in vivo CLL microenvironment. Results: Forty CLL primary patient PBMC samples, selected to represent diverse prognostic subgroups, were cultured in media supplemented with LEN, CC-122, or control and assessed in a BrdU incorporation assay. Among these analyzed samples, four distinct ‘response’ subsets were identified. In subset 1 (n=10, 25% of cases), we found that CC-122 is superior to LEN in inhibiting proliferation (mean IC50: LEN 5.7µM; CC-122 1.2µM). Subset 2 (n=23; 57% of cases) was sensitive to CC-122, but unresponsive to LEN. In these cases, CC-122 inhibited proliferation by at least 50% at a concentration of ≤3µM despite the failure of LEN (IC50: >10 µM) to inhibit proliferation, highlighting a qualitative difference between the two agents. In subset 3, the anti-proliferative activities of CC-122 and LEN were comparable (n=2, 5% of cases). Finally, both CC-122 and LEN were inactive (up to 10µM) in a 4th subset (n=5; 12% of cases). We subsequently explored associations between CC-122 activity and known CLL prognostic risk factors in these samples. In 5 out of 6 cases with 17p deletion CC-122 inhibited proliferation (IC50: Conclusions: Taken together, these data demonstrate that CC-122 has robust anti-proliferative activity in primary CLL cells through a unique mechanism of selective degradation of Aiolos and Ikaros transcription factors. CC-122 is superior to LEN and is active across several prognostic backgrounds. Furthermore, we show significant activity of CC-122 even in cases that are non-responsive to LEN suggesting that CC-122 targets a novel pathway in CLL. These data suggest that further evaluations of CC-122 in pre-clinical and clinical studies, as a single agent or in combination with established or novel compounds (such as Btk and PI3Kd inhibitors), are warranted. Disclosures Blocksidge: Celgene: Research Funding. Glenn:Celgene: Research Funding. Gandhi:Celgene Corp: Employment, Equity Ownership. Klippel:Celgene: Employment, Equity Ownership. Pourdehnad:Celgene: Employment, Equity Ownership. Chopra:Celgene Corp: Employment, Equity Ownership. Kalakonda:Celgene: Research Funding.