Preliminary analysis

[niranjchandrasekaran]

Goals: Exploratory analysis to ensure everything is working as expected and look for relationships between perturbations

Conclusion:

[niranjchandrasekaran]

Resources

• Information about this project can be found on ipprojects (Project tag: ipprojects+p_18kinase@broadinstitute.org)
• Additional information can be found on this sheet.

[niranjchandrasekaran]

Experimental details

• 11 perturbations x 3 doses (not all compounds have the same three concentrations; there are six different concentrations across all perturbations)
• 8 replicates per dose per perturbation
• 104 DMSO wells
• 4 positive controls x 4 replicates
• 4 cell types (PATU-8988T, MDA-MB-231, U2OS, A549)
• 2 replicate plates per cell type
• Experiment metadata

Platemap and metadata files are in the metadata folder.

Plate map visualization

Notebook

[niranjchandrasekaran]

Notebook

I calculated the replicate correlations and Percent Replicating for each cell type separately.

Percent Replicating

U2OS has the lowest scores while the other three have much higher scores. Each dose point is considered a separate sample

Description	Percent_Replicating
Cell Type=PATU-8988T	51.5
Cell Type=MDA-MB-231	57.6
Cell Type=U2OS	39.4
Cell Type=A549	66.7

Correlation distributions


Replicate Correlation

Table

| Cell_Type | Metadata_Supplier_Catalog | Concentration | replicate_correlation | |:------------|:----------------------------|----------------:|------------------------:| | PATU-8988T | AX15836 | 0.04 | 0.207598 | | PATU-8988T | AX15836 | 0.2 | 0.286918 | | PATU-8988T | AX15836 | 0.4 | 0.311544 | | PATU-8988T | BSJ-04-146 | 0.004 | 0.662566 | | PATU-8988T | BSJ-04-146 | 0.01 | 0.746326 | | PATU-8988T | BSJ-04-146 | 0.04 | 0.788334 | | PATU-8988T | BSJ-04-175 | 0.004 | 0.396165 | | PATU-8988T | BSJ-04-175 | 0.01 | 0.380156 | | PATU-8988T | BSJ-04-175 | 0.04 | 0.500605 | | PATU-8988T | BSJ-05-002 | 0.004 | 0.261445 | | PATU-8988T | BSJ-05-002 | 0.01 | 0.231175 | | PATU-8988T | BSJ-05-002 | 0.04 | 0.196023 | | PATU-8988T | FMF-03-146-1 | 0.2 | 0.286181 | | PATU-8988T | FMF-03-146-1 | 0.4 | 0.183296 | | PATU-8988T | FMF-03-146-1 | 1 | 0.262911 | | PATU-8988T | FMF-04-112-1 | 0.2 | 0.213167 | | PATU-8988T | FMF-04-112-1 | 0.4 | 0.115175 | | PATU-8988T | FMF-04-112-1 | 1 | 0.205731 | | PATU-8988T | FMF-04-159-2 | 0.04 | 0.788443 | | PATU-8988T | FMF-04-159-2 | 0.2 | 0.930483 | | PATU-8988T | FMF-04-159-2 | 0.4 | 0.945025 | | PATU-8988T | FMF-05-176-1 | 0.04 | 0.767895 | | PATU-8988T | FMF-05-176-1 | 0.2 | 0.936877 | | PATU-8988T | FMF-05-176-1 | 0.4 | 0.952607 | | PATU-8988T | JWG-071 | 0.04 | 0.189251 | | PATU-8988T | JWG-071 | 0.2 | 0.565885 | | PATU-8988T | JWG-071 | 0.4 | 0.634787 | | PATU-8988T | JWG-119 | 0.04 | 0.313824 | | PATU-8988T | JWG-119 | 0.2 | 0.564364 | | PATU-8988T | JWG-119 | 0.4 | 0.849087 | | PATU-8988T | Pin1-3 | 0.2 | 0.215287 | | PATU-8988T | Pin1-3 | 0.4 | 0.436452 | | PATU-8988T | Pin1-3 | 1 | 0.51543 | | MDA-MB-231 | AX15836 | 0.04 | 0.146829 | | MDA-MB-231 | AX15836 | 0.2 | 0.0641287 | | MDA-MB-231 | AX15836 | 0.4 | 0.23569 | | MDA-MB-231 | BSJ-04-146 | 0.004 | 0.763183 | | MDA-MB-231 | BSJ-04-146 | 0.01 | 0.757047 | | MDA-MB-231 | BSJ-04-146 | 0.04 | 0.80001 | | MDA-MB-231 | BSJ-04-175 | 0.004 | 0.111148 | | MDA-MB-231 | BSJ-04-175 | 0.01 | 0.210215 | | MDA-MB-231 | BSJ-04-175 | 0.04 | 0.192491 | | MDA-MB-231 | BSJ-05-002 | 0.004 | 0.0857121 | | MDA-MB-231 | BSJ-05-002 | 0.01 | 0.231145 | | MDA-MB-231 | BSJ-05-002 | 0.04 | 0.212839 | | MDA-MB-231 | FMF-03-146-1 | 0.2 | 0.194628 | | MDA-MB-231 | FMF-03-146-1 | 0.4 | 0.424091 | | MDA-MB-231 | FMF-03-146-1 | 1 | 0.856013 | | MDA-MB-231 | FMF-04-112-1 | 0.2 | 0.1463 | | MDA-MB-231 | FMF-04-112-1 | 0.4 | 0.458296 | | MDA-MB-231 | FMF-04-112-1 | 1 | 0.89886 | | MDA-MB-231 | FMF-04-159-2 | 0.04 | 0.374882 | | MDA-MB-231 | FMF-04-159-2 | 0.2 | 0.892834 | | MDA-MB-231 | FMF-04-159-2 | 0.4 | 0.995991 | | MDA-MB-231 | FMF-05-176-1 | 0.04 | 0.343722 | | MDA-MB-231 | FMF-05-176-1 | 0.2 | 0.822603 | | MDA-MB-231 | FMF-05-176-1 | 0.4 | 0.903412 | | MDA-MB-231 | JWG-071 | 0.04 | 0.0985972 | | MDA-MB-231 | JWG-071 | 0.2 | 0.41292 | | MDA-MB-231 | JWG-071 | 0.4 | 0.61846 | | MDA-MB-231 | JWG-119 | 0.04 | 0.46331 | | MDA-MB-231 | JWG-119 | 0.2 | 0.767557 | | MDA-MB-231 | JWG-119 | 0.4 | 0.916923 | | MDA-MB-231 | Pin1-3 | 0.2 | 0.134575 | | MDA-MB-231 | Pin1-3 | 0.4 | 0.205606 | | MDA-MB-231 | Pin1-3 | 1 | 0.467378 | | U2OS | AX15836 | 0.04 | 0.0962827 | | U2OS | AX15836 | 0.2 | 0.142485 | | U2OS | AX15836 | 0.4 | 0.133493 | | U2OS | BSJ-04-146 | 0.004 | 0.186816 | | U2OS | BSJ-04-146 | 0.01 | 0.285566 | | U2OS | BSJ-04-146 | 0.04 | 0.302382 | | U2OS | BSJ-04-175 | 0.004 | 0.10615 | | U2OS | BSJ-04-175 | 0.01 | 0.127351 | | U2OS | BSJ-04-175 | 0.04 | 0.109914 | | U2OS | BSJ-05-002 | 0.004 | 0.117049 | | U2OS | BSJ-05-002 | 0.01 | 0.193093 | | U2OS | BSJ-05-002 | 0.04 | 0.0997503 | | U2OS | FMF-03-146-1 | 0.2 | 0.190366 | | U2OS | FMF-03-146-1 | 0.4 | 0.109527 | | U2OS | FMF-03-146-1 | 1 | 0.142722 | | U2OS | FMF-04-112-1 | 0.2 | 0.126925 | | U2OS | FMF-04-112-1 | 0.4 | 0.129365 | | U2OS | FMF-04-112-1 | 1 | 0.206239 | | U2OS | FMF-04-159-2 | 0.04 | 0.140323 | | U2OS | FMF-04-159-2 | 0.2 | 0.244941 | | U2OS | FMF-04-159-2 | 0.4 | 0.509743 | | U2OS | FMF-05-176-1 | 0.04 | 0.0686473 | | U2OS | FMF-05-176-1 | 0.2 | 0.138731 | | U2OS | FMF-05-176-1 | 0.4 | 0.137602 | | U2OS | JWG-071 | 0.04 | 0.143943 | | U2OS | JWG-071 | 0.2 | 0.209039 | | U2OS | JWG-071 | 0.4 | 0.278895 | | U2OS | JWG-119 | 0.04 | 0.138213 | | U2OS | JWG-119 | 0.2 | 0.347487 | | U2OS | JWG-119 | 0.4 | 0.664056 | | U2OS | Pin1-3 | 0.2 | 0.139628 | | U2OS | Pin1-3 | 0.4 | 0.129191 | | U2OS | Pin1-3 | 1 | 0.250375 | | A549 | AX15836 | 0.04 | 0.222148 | | A549 | AX15836 | 0.2 | 0.294647 | | A549 | AX15836 | 0.4 | 0.279722 | | A549 | BSJ-04-146 | 0.004 | 0.680998 | | A549 | BSJ-04-146 | 0.01 | 0.663322 | | A549 | BSJ-04-146 | 0.04 | 0.662048 | | A549 | BSJ-04-175 | 0.004 | 0.264287 | | A549 | BSJ-04-175 | 0.01 | 0.287224 | | A549 | BSJ-04-175 | 0.04 | 0.332538 | | A549 | BSJ-05-002 | 0.004 | 0.25896 | | A549 | BSJ-05-002 | 0.01 | 0.261305 | | A549 | BSJ-05-002 | 0.04 | 0.246232 | | A549 | FMF-03-146-1 | 0.2 | 0.275763 | | A549 | FMF-03-146-1 | 0.4 | 0.34513 | | A549 | FMF-03-146-1 | 1 | 0.404396 | | A549 | FMF-04-112-1 | 0.2 | 0.20369 | | A549 | FMF-04-112-1 | 0.4 | 0.335719 | | A549 | FMF-04-112-1 | 1 | 0.450231 | | A549 | FMF-04-159-2 | 0.04 | 0.351614 | | A549 | FMF-04-159-2 | 0.2 | 0.836304 | | A549 | FMF-04-159-2 | 0.4 | 0.796195 | | A549 | FMF-05-176-1 | 0.04 | 0.530787 | | A549 | FMF-05-176-1 | 0.2 | 0.912831 | | A549 | FMF-05-176-1 | 0.4 | 0.954327 | | A549 | JWG-071 | 0.04 | 0.221222 | | A549 | JWG-071 | 0.2 | 0.284907 | | A549 | JWG-071 | 0.4 | 0.425375 | | A549 | JWG-119 | 0.04 | 0.424629 | | A549 | JWG-119 | 0.2 | 0.581168 | | A549 | JWG-119 | 0.4 | 0.769701 | | A549 | Pin1-3 | 0.2 | 0.19297 | | A549 | Pin1-3 | 0.4 | 0.299867 | | A549 | Pin1-3 | 1 | 0.405832 |

I plotted replicate correlations as bar plots.

In order to be able to interpret whether a replicate correlation value is significantly different from the null distribution, here are the null thresholds

Description	Value_95
Cell Type=PATU-8988T	0.383839
Cell Type=MDA-MB-231	0.305252
Cell Type=U2OS	0.177008
Cell Type=A549	0.285751

The following are the MOA of the compounds which will perhaps help interpret the results

Supplier_Catalog	moa
FMF-03-146-1	DCLK1 inhibitor
FMF-04-112-1	DCLK1 negative control
FMF-04-159-2	TAIRE kinase inhibitor covalent
FMF-05-176-1	TAIRE kinase inhibitor reversible control
BSJ-04-146	undisclosed target PROTAC
BSJ-05-002	undisclosed target PROTAC negative control
BSJ-04-175	undisclosed target parental inhibitor
Pin1-3	Pin1 inhibitor covalent
JWG-071	ERK5 inhibitor
AX15836	ERK5 inhibitor
JWG-119	ERK5 inhibitor negative control

Grouped by Concentration

Replicate correlation seems to increase with concentration, which is good. A couple of other observations I made are as follows

1. The undisclosed target PROTAC (BSJ-04-146) and undisclosed target PROTAC negative control (BSJ-05-002) have high and low replicate correlation values, respectively.
2. The ERK5 inhibitor negative control (JWG-119) and ERK5 inhibitor (AX15836) have high and low replicate correlation values, respectively.

Grouped by Cell type

As I mentioned above, these compounds don't seem to be very active in U2OS.

Stacked plots

I stacked the bars so that overall effect of the compounds across all cell concentrations and cell types can be visualized.

Morpheus

I used the replicate collapsed gct file to create this morpheus save session. I grouped the heatmap by cell type. There are number of interesting blue areas all over the heatmap. In general, they seem to correspond to the BSJ or FMF compounds.

[niranjchandrasekaran]

cc @AnneCarpenter

[niranjchandrasekaran]

I added all the controls and the updated gct file and morpheus session are here and here respectively.

I tried using euclidean distance as the similarity metric for clustering. I clipped the values at 200 so that all the shades of red are visible. I am not sure if this heatmap is more informative than the one above.

[AnneCarpenter]

Thanks! I agree that adding DMSO to these plots, when it's been normalized almost to oblivion and has a very low replicate correlation is not very helpful because it could easily land in any of the clusters, just by slightly changes in the profile relative to 0.