Akt is activated in a subset of primary MM samples and this determines their vulnerability to pharmacologic Akt inhibition. (A-C) IHC, phospho-Akt–specific flow cytometry, and susceptibility to pharmacologic Akt inhibition in 2 exemplary primary MM samples. (Ai,ii) Immunohistochemical detection of phospho-Akt in bone marrow biopsies heavily infiltrated by MM cells (right panels). Giemsa stained overview and IHC staining for plasma-cell marker CD138 (inset) shown to the left. Original magnifications, 400× (left) and 200× (right and insets). (Bi,ii) Phospho-Akt–specific flow cytometry in freshly purified CD138⁺ MM cells from the same patients as in panel A. A total of 50 000 cells per well were kept for a total of 24 hours in medium alone. They were then treated overnight with Akti-1/2 (10 μM) or DMSO. (Ci,ii) Viability of freshly purified CD138⁺ MM cells from the same patients as in panels A and B after treatment with Akti-1/2 for 5 days. Cells were cocultured with BMSCs and survival was determined by annexin V–FITC/PI staining and flow cytometry. The percentage of viable cells reflects comparison to DMSO-treated controls. Of note, whereas the Akti-1/2 resistant sample (patient 17) did not display Akt phosphorylation, the Akti-1/2–sensitive case (patient 6) showed strong phospho-Akt signals. (D) Effect plot of all primary MM samples tested (n = 30) for their susceptibility to treatment with 10 μM Akti-1/2. Viability determined as described in panel C. The medians for the strongly sensitive and the more resilient/resistant groups are indicated. (E) Correlation of Akt activation in BM biopsies with viability of MM cells from the corresponding patient after treatment with Akti-1/2 (n = 19). Viability was determined as described in panel C. (F) Correlation of the fold change in MFI for phospho-Akt in primary MM cells with their viability after treatment with Akti-1/2 (n = 20). Measurements performed as described in panels B and C. Horizontal bars mark the medians.