Plan for the next months

New experiments relevant for the paper (ranked for priority):

 Get TSA values from Jelena
 Design of mutants which revert the cooperative effect between Aurora A and
Cereblon (Matthias/Bikash)
 si/sh Aurora A: BrdU/PI FACS (Bikash) , also look into the literature: did
people deplete and performed Annexin/PI FACS?
 Biological effect in other cell lines: Test of lethality (crystal violet?(Bikash),
Alamar blue? (Markus/Mahmoud)) and Apoptosis (annexin/PI (Bikash)) after
long JB treatment
 Look at stress sensors after JB (positive control:UV): pATR, pATM, pCheck,
RPA, yH2X, MVM-proteins etc. (you have to ask Eilers people for their
experience with the antibodies: Steffi, Gabriele, Theresa) (Bikash)
 Co-IPs between Cereblon and Aurora: +/-JB, MG132 (Bikash)
 Small Synergisms screen(UV, Chemo, ?) (lets discuss this with
Markus/Mahmod)
 Aurora-HiBit-measurement: time coarse for early time points (Bikash); or:
express largeBit and measure a time course in vivo (Markus/Mahmod)
 RT PCR for some G2M genes after Alisertib and JB treatment, are their genes
indicative for JB and not Alisertib? (Bikash)
 Deplete Cereblon with sgRNAs, Protac should be non-functional, can the cells
(which ones?) live without Cereblon? Are their Cancer cells depended on
Cereblon. (sg-cloning and infection: Jessy, Analysis: Bikash)
 Deplete Cereblon, do AuroraA levels go up? (is Cereblon a natural ligase for
Aurora?)
 Does degradation work in mouse cells (KPCs, 3T3s) as well? (Bikash)
 Try rescue with kinase-dead mutant (Jessy can clone)
 JB effect in non-transformed cells (Markus)
 Aurora-A levels and IF in S-phase (Apoorva)?

Analysis of existing experiments

 RNA-seq, 4sU-seq. general analysis. Identification of genes, which are
indicative for JB (unknown, play a role in S-phase checkpoint UV?) and
Alisertib (G2M), (Apoorva, Bikash, Elmar)
 Mass spec: SILAC in MV411 (e.g. Vulcano, intensity-Plot, double-log2-FC-
Plot) (Elmar/Bikash/Andreas)
 Other SILAC MS? Other MS?
 What else?
Replicates
 Repeat all important experiments to reach n=3 (if possible), e.g. all Apoptosis
experiments, the EC50 estimation (HiBit)

Unbiased experiments for mechanism:

 Aurora Interactome: +/- synchronization, +/- RNAse (Julia)
 Gradient centrifugation of Aurora +/- RNAse (?)
 Nucleotide levels after JB (Bikash/Werner)
 Chromatin MS, maybe optimize fractionation, find a marker for nucleoplasm,
stain MYC in the old experiments (Bikash, ask others for advise)
 IF of Aurora A in S-phase? (Aporva/Pranjali)
Important for follow ups:
 Optimze the linker, Alisertib, thalidomide and the connection points based on
the modeling, make the drug more hydrophobic (Mathias)
 Expression plasmid for Aurora A (Richard?), Co-crystallization with Cereblon
and JB170, aim: conformation of the modeling, PROTAC optimization
(Lars/Julia/Bikash/Matthias/Elmar)
 Big Synergisms screen (Martin Sos, Mathias Rosenfeld, assay:
Markus/Muschler)
 Measure degradation products of JB after incubation, also as a quality check
for new synthesis (Bikash/Werner)
 Plan MV4-11 xenografts, look up, if people did this (Nevenka)
 Protacs with other (not patented) Aurora A Inhibitors (contact of Martin Sos,
Med Chem guy) and un-protected Thalidomide (check with med Chem guy)
mit anderen (am liebsten ungeschützten)
 Identify entities, which are especially dependent on Aurora (Broad/Zuber drop
out screens, Elmar)
 Check mutation status of Aurora A in various entities (Silke, Elmar)
 Measure cell permeability with Caco assay, make linker more hydrophobic
(Nevenka)
 Measure “in vivo stability” in vitro: liver mikrosomes? (Nevenka/Steffi Peter)

Not sure if necessary::

 MS with JB 211
 MLN4924 instead of MG?
 Cycloheximide (Jessy?)
 DC50 in Alamar-blue assays?

Für Stefan:
 Cereblon Expression zeigen
 Make linker more hydrophobic

Mathias Diebold
 Optimized linker (length and hydrophobixity)
 Optimized Alisertib and thalidomide
 Interface mutations
 Modeling with AuroraB
