Anti-Myeloma Activity of Akt Inhibition Is Linked to the Activation Status of PI3K/Akt and MEK/ERK Pathway

<div><p>The PI3K/Akt/mTOR signal transduction pathway plays a central role in multiple myeloma (MM) disease progression and development of therapeutic resistance. mTORC1 inhibitors have shown limited efficacy in the clinic, largely attributed to the reactivation of Akt due to rapamycin induced mTORC2 activity. Here, we present promising anti-myeloma activity of MK-2206, a novel allosteric pan-Akt inhibitor, in MM cell lines and patient cells. MK-2206 was able to induce cytotoxicity and inhibit proliferation in all MM cell lines tested, albeit with significant heterogeneity that was highly dependent on basal pAkt levels. MK-2206 was able to inhibit proliferation of MM cells even when cultured with marrow stromal cells or tumor promoting cytokines. The induction of cytotoxicity was due to apoptosis, which at least partially was mediated by caspases. MK-2206 inhibited pAkt and its down-stream targets and up-regulated pErk in MM cells. Using MK-2206 in combination with rapamycin (mTORC1 inhibitor), LY294002 (PI3K inhibitor), or U0126 (MEK1/2 inhibitor), we show that Erk- mediated downstream activation of PI3K/Akt pathway results in resistance to Akt inhibition. These provide the basis for clinical evaluation of MK-2206 alone or in combination in MM and potential use of baseline pAkt and pErk as biomarkers for patient selection.</p> </div

MK-2206 induced apoptosis in MM cell lines and patient cells.

<p>We incubated MM1S and OPM2 cells with <b>A)</b> indicated concentrations of MK-2206 for 48 hrs and <b>B)</b> with 2.5 µM of MK-2206 for 12, 24 or 48 hrs. We observed dose and time dependent increase in apoptosis as measured by annexin/PI staining. % cells in each quadrant is indicated. <b>C)</b> MM1S or <b>D)</b> OPM2 with indicated concentrations of MK-2206 for 48 hrs and measured activation of caspases 9, 8 and 3. We observed dose dependent increase in activation of each of the three caspases. In all the above experiments, control refers to cells untreated with MK-2206. We incubated <b>E)</b> MM1S or <b>F)</b> OPM2 cells with indicated concentrations of MK-2206, MK-2206 plus Q-VD-OPH (10 µM), MK-2206 plus Z-IETD-FMK (10 µM) or MK-2206 plus Ac-LEHD-CMK(10 µM) for 48 hrs. We observed dose dependent decrease in cell viability, which was partially blocked by Q-VD-OPH. MK-2206 concentration is indicated along×axis and viability (% of control) is indicated along Y-axis. Error bars represent standard deviation. <b>G)</b> We incubated cells from MM patients with indicated concentrations of MK-2206 for 48 hrs. We then measured for plasma cells undergoing apoptosis by annexin/PI staining. MK-2206 concentration is indicated along×axis and viability (% of control) is indicated along Y axis.</p

MK-2206 induced cytotoxicity, inhibited proliferation and was able to overcome the protective effects of the tumor microenvironment.

<p>When we cultured MM cell lines with indicated concentrations of MK-2206 for 48 hrs we observed dose dependent decrease in <b>A)</b> viability as observed by MTT assays and <b>B)</b> tritiated thymidine uptake indicative of inhibition of proliferation albeit with differences in the sensitivity of cell lines to MK-2206. MK-2206 concentration is indicated along the×axis and in <b>A)</b> viability is expressed as % of control is indicated along the Y axis and in B) proliferation is expressed as % cpm of control. Error bars represent standard deviation. <b>C)</b> We evaluated baseline levels of activated Akt and down-stream members of the PI3K/Akt/mTOR pathway including pmTOR, p-p70S6K, p4EBP1 and in addition pErk levels by western blotting. We observed that cell lines sensitive to MK-2206 expressed high levels of pAkt whereas the cell lines less sensitive to MK-2206 expressed low levels of pAkt but high levels of pErk. When MM1S cells were co-cultured with <b>D)</b> bone marrow stromal cells (BMSCs) or <b>E)</b> cytokines IL6 (25 ng/ml), IGF (50 ng/ml) or VEGF (50 ng/ml), we observed increase in proliferation measured by increase in cpm. MK-2206 was able to inhibit this increase in proliferation. MK-2206 concentrations are indicated on X-axis and % cpm is indicated on the Y-axis. Error bars represent standard deviation. <b>F)</b> We cultured MM1S cells with or without 2.5 µM of MK-2206 for 8 hrs. In the last thirty minutes of incubation, cells were treated with IGF (50 ng/ml) for indicated time points. We then made lysates and checked for the ability of MK-2206 to inhibit pAkt and down-stream members of the PI3K/Akt/mTOR pathway. We observed potent down regulation of pAkt and downstream members with no differences observed in total proteins and actin, which serve as loading controls.</p

MK-2206 inhibits pAkt and downstream members of the PI3K/Akt/mTOR pathway and modulates expression levels of other signaling pathway proteins.

<p>We incubated <b>A and E)</b> MM1S or <b>B and F)</b> OPM2 cells with 2.5 µM of MK-2206 for 1, 2, 4 or 8 hrs. We observed down regulation of pAkt and downstream members of the pathway in both MM1S and OPM2 cells. We observed up regulation of pErk in both cell lines. We also observed down regulation of pBad and Bcl-Xl. No differences were observed in total proteins and actin, which serve as loading controls. We incubated CD138+ cells from <b>C and G)</b> Patient 1 or <b>D and H)</b> Patient 2 with 2.5 µM of MK-2206 for indicated time points and observed down regulation of pAkt, pmTOR, p-p70S6K, p4EBP1 and pGsk3β. We observed down regulation of Mcl1 and Xiap levels in patient 1 and down regulation of Mcl1 in patient 2. In all the above experiments control refers to cells untreated with MK-2206.</p

MK-2206 synergizes with other inhibitors of the PI3K/Akt/mTOR pathway.

<p>When MK-2206 was combined with the mTOR inhibitor rapamycin, synergistic cytotoxicity was observed <b>A)</b> in cell lines sensitive to MK-2206 (MM1S, MM1R and OPM2) and <b>B)</b> in cell lines less sensitive to MK-2206 (RPMI8226, DOX40 and U266) as a single agent as measured using the MTT assay. The graph represents the concentrations of MK-2206, rapamycin or the combination at which maximum synergy was observed. The concentrations are: 1) MM1S−0.5 µM MK-2206, 5 nM rapamycin, 2) MM1R−0.5 µM MK-2206, 5 nM rapamycin and 3) OPM2−0.5 µM MK-2206, 5 nM rapamycin. 4) RPMI8226−2.5 µM MK-2206, 20 nM rapamycin, 5) DOX40−2.5 µM MK-2206, 5 nM rapamycin and 6) U266−5 µM MK-2206, 10 nM rapamycin. Cell lines used are indicated on the X-axis and Viability (% of control) indicated on the Y-axis. Error bars represent standard deviation. <b>C)</b> When MK-2206 was combined with the PI3K inhibitor LY294002, synergistic cytotoxicity was observed in cell lines sensitive to MK-2206 (MM1S and OPM2) as a single agent as measured using the MTT assay. The graph represents the concentrations of MK-2206, LY294002 or the combination at which maximum synergy was observed. The concentrations are: 1) MM1S−0.5 µM MK-2206, 10 µM LY294002 and 2) OPM2−0.5 µM MK-2206, 10 µM LY294002. Cell lines used are indicated on the X-axis and Viability (% of control) indicated on the Y-axis. Error bars represent standard deviation.</p

MK-2206 synergizes with the Mek inhibitor U0126 and the drug combination leads to increased inhibition of mTOR activity.

<p>When MK-2206 was combined with the Mek1/2 inhibitor U0126, synergistic cytotoxicity was observed <b>A)</b> in cell lines sensitive to MK-2206 (MM1S, MM1R and OPM2) and <b>B)</b> in cell lines less sensitive to MK-2206 (RPMI8226, DOX40 and U266) as a single agent as measured using the MTT assay. The graph represents the concentrations of MK-2206, U0126 or the combination at which maximum synergy was observed. The concentrations are: 1) MM1S−0.5 µM MK-2206, 10 µM U0126, 2) MM1R−0.5 µM MK-2206, 10 µM U0126 and 3) OPM2−1 µM MK-2206, 20 µM U0126. 4) RPMI8226−2.5 µM MK-2206, 10 µM U0126, 5) DOX40−5 µM MK-2206, 20 µM U0126 and 6) U266−5 µM MK-2206, 30 µM U0126. Cell lines used are indicated on the X-axis and Viability (% of control) indicated on the Y-axis. Error bars represent standard deviation. <b>C)</b> MM1S cells were left untreated or treated with U0126 (10 µM) for 24 hrs. Following this, cells were treated with 2.5 µM of MK-2206 for 1, 2, 4 or 8 hrs. As a control, MM1S cells were treated with U0126 alone (10 µM) for 32 hrs or MK-2206 alone (2.5 µM) for 8 hrs. We observed significant down regulation of pAkt and other downstream members of the pathway and pErk when MK-2206 was used in combination with U0126 with no difference in total proteins and actin, which serve as loading controls.</p

DataSheet_1_Nivolumab and daratumumab combination regimens for the treatment of relapsed and refractory multiple myeloma: results of a randomized phase I/II clinical trial.docx

PurposeThe phase I/II trial CheckMate 039 (NCT01592370) evaluated the safety, tolerability, and efficacy of nivolumab-daratumumab (ND) in patients with relapsed/refractory multiple myeloma (RRMM).MethodsPatients with RRMM were randomized to receive ND with or without pomalidomide-dexamethasone (Pd) in cohort A and ND or D monotherapy in cohort B. The primary endpoint was safety. Secondary endpoints included minimal residual disease (MRD) negativity status, overall response rate (ORR), duration of response, and progression-free survival (PFS).ResultsCohort A (n = 11) was terminated early due to safety concerns observed with immunomodulatory agents and checkpoint inhibitor combinations in other clinical trials. In the small number of patients treated in cohort A, no new safety concerns were observed but patients who received NDPd had numerically more grade 3/4 adverse events (AEs) and serious AEs compared with ND. Grade 3/4 AEs occurring in ≥ 1 patient in the ND group was anemia (3/6 patients); in the NDPd group, these were neutropenia (3/5 patients), upper respiratory tract infection (2/6 patients), and pneumonia (2/6 patients). In cohort B (n = 63), AE rates were similar between ND and D (any-grade: 87.8% vs 95.5%; grade 3/4: 53.7% vs 45.5%). Grade 3/4 AEs occurring in ≥ 1 patient in the ND group were neutropenia (19.5%), anemia (9.8%), thrombocytopenia (9.8%), and bronchitis (7.3%); in the D group these were anemia and pneumonia (both 9.1%). Immune-mediated AEs for ND were consistent with the known safety profile of nivolumab. In cohort A, all patients (5/5) receiving NDPd and 4/6 receiving ND achieved a response. In cohort B, the ORR with ND was numerically higher than D (22/41 [53.7%] vs 9/22 [40.9%]) and both groups had a median PFS of 6.6 months. ND also showed promising MRD negativity results (next-generation sequencing 10-5, 24.0%; next-generation flow 10-5, 22.2%).ConclusionNDPd demonstrated no new safety signals and encouraging efficacy despite its early termination. ND was well tolerated with a manageable toxicity and few AEs leading to discontinuation, and demonstrated a numerically higher ORR but equivalent PFS compared with D. Any clinical benefits to OS require a longer follow-up.Clinical trial registrationhttps://clinicaltrials.gov/, identifier NCT01592370.</p

Adequacy of the gamma distribution.

<p>The gamma distribution provides an adequate fit for multiple types of pedigrees. For example, HRP UT-549917 has <i>k</i> = 4.4 and <i>σ</i> = 3.6 with good visual density (a) and CDF (b) fit, with <i>λ</i> = 0.9. (Goodness of fit was estimated with <i>λ</i>, the median of empirical chi-squared distribution divided by the median of the expected chi-squared distribution.) HRP UT-34955 has <i>k</i> = 2.8 and <i>σ</i> = 2.9 with good visual density (c) and CDF (d) fit, with <i>λ</i> = 1.0.</p

Significant or overlapping SGSs and segregating SNVs.

<p>Significant or overlapping SGSs and segregating SNVs.</p

Adequacy of the gamma distribution.

<p>The gamma distribution provides an adequate fit for multiple types of pedigrees. For example, HRP UT-549917 has <i>k</i> = 4.4 and <i>σ</i> = 3.6 with good visual density (a) and CDF (b) fit, with <i>λ</i> = 0.9. (Goodness of fit was estimated with <i>λ</i>, the median of empirical chi-squared distribution divided by the median of the expected chi-squared distribution.) HRP UT-34955 has <i>k</i> = 2.8 and <i>σ</i> = 2.9 with good visual density (c) and CDF (d) fit, with <i>λ</i> = 1.0.</p