Although in vivo targeting of tumors using fluorescently labeled probes has greatly gained in importance over the last few years, most of the clinically applied reagents lack tumor cell specificity. Our novel tumor cell-penetrating peptide-based probe (TPP) recognizes an epitope of Hsp70 that is exclusively present on the cell surface of a broad variety of human and mouse tumors and metastases, but not on normal tissues. Because of the rapid turnover rate of membrane Hsp70, fluorescently labeled TPP is continuously internalized into syngeneic, spontaneous, chemically/genetically induced and xenograft tumors following intravenous administration, thereby enabling site-specific labeling of primary tumors and metastases. In contrast with the commercially available nonpeptide small molecule a v b 3 -integrin antagonist IntegriSense, TPP exhibits a significantly higher tumor-to-background contrast and stronger tumor-specific signal intensity in all tested tumor models. Moreover, in contrast with IntegriSense, TPP reliably differentiates between tumor cells and cells of the tumor microenvironment, such as tumor-associated macrophages and fibroblasts, which were found to be membrane-Hsp70 negative. Therefore, TPP provides a useful tool for multimodal imaging of tumors and metastases that might help to improve our understanding of tumorigenesis and allow the establishment of improved diagnostic procedures and more accurate therapeutic monitoring. TPP might also be a promising platform for tumor-specific drug delivery and other Hsp70-based targeted therapies. Cancer Res; 74(23); 6903-12. Ó2014 AACR.

Introduction: Mantle cell lymphoma (MCL) comprises about 6% of all non-Hodgkin's lymphoma with a median survival of 3-5 years. Constitutional activation of the mTOR/AKT pathway has been identified in the majority of cases (Rudelius, Blood 2006). The pro-viral insertion in murine (PIM) lymphoma proteins are serine/threonine kinases which play a critical role in cell survival as well as proliferation and identifies a high risk patient cohort with MCL (Hsi, Leuk Lymphoma 2008). In this study we evaluated the efficiency and mode of action of a dual PIM/PI3K (IBL-202) and a triple PIM/PI3K/mTOR inhibitor (IBL-301) in MCL cell lines and primary cells. Methods: MCL cell lines (Granta 519, Jeko-1, Rec-1 and Mino), as well as primary MCL cells were exposed to the combined PIM-kinase/PI3K (IBL-202) and the PIM-kinase/PI3K/mTOR Inhibitor (IBL-301). Cell proliferation (trypan blue staining), cell apoptosis (Annexin V PE/7-AAD staining) and cell cycle (FACS) were investigated. Protein expression and phosphorylation status of different downstream proteins (Akt, GSK-3β, 4EBP1) as well as markers of apoptosis (PARP, Caspase 9) were analysed after 1h, 4h, 8h and 24h. Cell viability was assessed by CellTiter-Glo® assay after 48h. Results: Both inhibitors led to G1 arrest. At 500 nM, the triple inhibitor IBL-301 (19,8%) is in average slightly more efficacious than the dual-inhibitor IBL-202 (13,5%). Accordingly, IBL-301 had a much higher impact on cell proliferation than IBL-202 in all tested MCL cell lines (reduction by 48 - 93% vs 22 - 87%), possibly due to its mTOR inhibitory potential, although it may be also a more potent inhibitor of PIM and PI3K kinases. In addition, treatment with IBL-202 and IBL-301 induced cell apoptosis in Jeko-1, Rec-1 and Mino. Again, rate of apoptosis by IBL-301 was much higher (e.g. JEKO: 56% vs 13%) and could be achieved at lower concentrations in comparison to IBL-202. The differential impact on apoptosis could be confirmed based on PARP and Caspase 9 cleavage, which was higher after treatment with IBL-301 after 24h. In Jeko-1, Granta-519 and Mino both agents led to de-phosphorylation of Akt. Interestingly, this effect was more prominent in IBL-301 treated cell lines, supporting the mode of action via the PI3K-AKT pathway of both inhibitors. De-phosphorylation of GSK-3β was observed in all tested MCL cell lines with both inhibitors already during the first hour of exposure and was reversible thereafter. Primary MCL cells of 2 patients were treated with 62.5 nM IBL-202, 31.25 nM IBL-301 and single inhibitors of PIM (2.5 µM AZD1208), PI3K (1.25 µM idelalisib) and mTOR (5 nM temsirolimus). Viability after 48h was reduced by about 70% following IBL-301 exposure compared to 39% in IBL-202 treated samples. Both combined inhibitors were more potent than any of the single inhibitors. IBL-301 and IBL-202 decreased viability in a similar way as the combination of AZD1208, idelalisib +/- temsirolimus. Normal lymphocytes tolerated both inhibitors in various concentrations (62,5 - 500 nM). Conclusions: Triple inhibition of PIM kinases, PI3K and mTOR is very efficient in MCL cell lines as well as in primary MCL cells, exceeding dual inhibition of PIM kinases and PI3K. Thus, cotargeting PIM kinases, PI3K and mTOR may be a promising novel approach for clinical development in MCL. Disclosures O'Neill: Inflection Biosciences: Employment.

Introduction: Mantle cell lymphoma (MCL) is characterized by t(11;14) resulting in a constitutive cyclin D1 overexpression. The cyclin D1-CDK4/6 complex inactivates Rb through phosphorylation, leading to G1/S-phase transition. Therefore, inhibition of CDK4/6 is an efficient and rational approach to overcome cell cycle dysregulation in MCL. We evaluated the efficiency of the novel CDK4/6 inhibitor abemaciclib in various MCL cell lines and in primary MCL cells in combination with cytarabine (AraC) and ibrutinib. Material & Methods: MCL cell lines (Granta 519, JeKo-1, Maver-1, Mino) and primary MCL cells were exposed to abemaciclib alone and combined with AraC or ibrutinib. Cells were pretreated with abemaciclib and exposed to AraC or ibrutinib with or without consecutive wash-out of the CDK4/6 inhibitor. Proliferation and viability were measured by tryptan blue staining and Cell Titer Glo assay. Flow cytometry was used for cell-cycle (PI-staining) and apoptosis analysis (Annexin V PE/7AAD-staining). Western Blot analysis showed protein expression and phosphorylation status of various downstream proteins. Results: Abemaciclib inhibited cell proliferation by induction of early G1-arrest. Western Blot analysis revealed reduced phosphorylation of Rb on serine 795 without changes in CDK 4 and cyclin D1 expression, in line with reversible cell cycle arrest. IC50-values of sensitive cell lines (JeKo-1, Maver-1, Mino) were <30 nM after 72 h. We observed an almost complete and reversible G1-arrest in all sensitive cell lines by FACS analysis (JeKo-1: G1-phase +51,7 %; S/G2-phase -51,7 % at 31,25 nM after 24 h; G1-phase +35,4 %; S/G2-phase -34,8 % after 72 h), whereas cell viability was not reduced. Wash-out of abemaciclib after 24 h resulted in synchronized S-phase entry in all sensitive cell lines (e.g. Mino: G1-phase -20,4 %; S-phase +30,5 %). The sequential combination of abemaciclib followed by AraC showed strong synergy in Mino cells (CI=0,22 for 31,25 nM abemaciclib and 3,33 µM cytarabine). In contrast, simultaneous exposure to abemaciclib had a protective effect against AraC treatment in all sensitive cell lines, due to an ongoing G1-arrest (Mino: CI=-0,19 for 31,25 nM abemaciclib and 3,33 µM AraC). In primary MCL cells, 31,25 nM of abemaciclib had no impact on cell death. Moreover, no sensitization to AraC was observed as all cells were resting in G0-phase. The combination of abemaciclib induced G1 arrest and ibrutinib had additive or synergistic effects in sensitive cell lines (JeKo-1, Mino and Maver). Conclusion: The novel CDK4/6 inhibitor abemaciclib causes reversible G1 cell cycle arrest without loss of viability at low nanomolar doses. Rationale drug combinations exploiting the sequential effect may achieve major benefits, but drug interactions are complex: Pretreatment with abemaciclib sensitizes MCL cell line cells to AraC whereas simultaneous application protects them from AraC treatment. Further analyses explore the interaction with other targeted approaches (inhibitors of the B-cell receptor pathway) to better understand the underlying molecular mechanisms. Disclosures No relevant conflicts of interest to declare.

<p>Supplemental Methods, Supplemental Figure legends Figure S1, Figure S2, Figure S3, Figure S4, Figure S5, Figure S6, Figure S7</p>