Multiple myeloma is highly dependent on the bone marrow microenvironment until progressing to very advanced extramedullary stages of the disease such as plasma cell leukemia. Stromal cells in the bone marrow secrete a variety of cytokines that promote plasma cell survival by regulating antiapoptotic members of the Bcl-2 family including Mcl-1, Bcl-x, and Bcl-2. Although the antiapoptotic protein on which a cell depends is typically consistent among normal cells of a particular phenotype, Bcl-2 family dependence is highly heterogeneous in multiple myeloma. Although normal plasma cells and most multiple myeloma cells require Mcl-1 for survival, a subset of myeloma is codependent on Bcl-2 and/or Bcl-x We investigated the role of the bone marrow microenvironment in determining Bcl-2 family dependence in multiple myeloma. We used the Bcl-2/Bcl-x inhibitor ABT-737 to study the factors regulating whether myeloma is Mcl-1 dependent, and thus resistant to ABT-737-induced apoptosis, or Bcl-2/Bcl-x codependent, and thus sensitive to ABT-737. We demonstrate that bone marrow stroma is capable of inducing Mcl-1 dependence through the production of the plasma cell survival cytokine interleukin-6 (IL-6). IL-6 upregulates Mcl-1 transcription in a STAT3-dependent manner, although this occurred in a minority of the cells tested. In all cells, IL-6 treatment results in posttranslational modification of the proapoptotic protein Bim. Phosphorylation of Bim shifts its binding from Bcl-2 and Bcl-x to Mcl-1, an effect reversed by MEK inhibition. Blocking IL-6 or downstream signaling restored Bcl-2/Bcl-x dependence and may therefore represent a clinically useful strategy to enhance the activity of Bcl-2 inhibitors.

Background Antibody drug conjugates (ADCs) targeting the epidermal growth factor receptor (EGFR), such as depatuxizumab mafodotin (Depatux-M), is a promising therapeutic strategy for glioblastoma (GBM) but recent clinical trials did not demonstrate a survival benefit. Understanding the mechanisms of failure for this promising strategy is critically important. Methods PDX models were employed to study efficacy of systemic vs intracranial delivery of Depatux-M. Immunofluorescence and MALDI-MSI were performed to detect drug levels in the brain. EGFR levels and compensatory pathways were studied using quantitative flow cytometry, Western blots, RNAseq, FISH and phosphoproteomics. Results Systemic delivery of Depatux-M was highly effective in nine of 10 EGFR-amplified heterotopic PDXs with survival extending beyond one year in eight PDXs. Acquired resistance in two PDXs (GBM12 and GBM46) was driven by suppression of EGFR expression or emergence of a novel short-variant of EGFR lacking the epitope for the Depatux-M antibody. In contrast to the profound benefit observed in heterotopic tumors, only two of seven intrinsically sensitive PDXs were responsive to Depatux-M as intracranial tumors. Poor efficacy in orthotopic PDXs was associated with limited and heterogeneous distribution of Depatux-M into tumor tissues, and artificial disruption of the BBB or bypass of the BBB by direct intracranial injection of Depatux-M into orthotopic tumors markedly enhanced the efficacy of drug treatment. Conclusions Despite profound intrinsic sensitivity to Depatux-M, limited drug delivery into brain tumor may have been a key contributor to lack of efficacy in recently failed clinical trials.

Engineered allosteric regulation of protein activity provides significant advantages for the development of robust and broadly applicable tools. However, the application of allosteric switches in optogenetics has been scarce and suffers from critical limitations. Here, we report an optogenetic approach that utilizes an engineered Light-Regulated (LightR) allosteric switch module to achieve tight spatiotemporal control of enzymatic activity. Using the tyrosine kinase Src as a model, we demonstrate efficient regulation of the kinase and identify temporally distinct signaling responses ranging from seconds to minutes. LightR-Src off-kinetics can be tuned by modulating the LightR photoconversion cycle. A fast cycling variant enables the stimulation of transient pulses and local regulation of activity in a selected region of a cell. The design of the LightR module ensures broad applicability of the tool, as we demonstrate by achieving light-mediated regulation of Abl and bRaf kinases as well as Cre recombinase.

Utilizing a protein carrier in combination with isobaric labeling to “boost” the signal of other low-level samples in multiplexed analyses has emerged as an attractive strategy to enhance data quantity while minimizing protein input in mass spectrometry analyses. Recent applications of this approach include pMHC profiling and tyrosine phosphoproteomics, two applications that are often limited by large sample requirements. While including a protein carrier has been shown to increase the number of identifiable peptides in both applications, the impact of a protein carrier on quantitative accuracy remains to be thoroughly explored, particularly in relevant biological contexts where samples exhibit dynamic changes in abundance across peptides. Here, we describe two sets of analyses comparing MS 2 -based quantitation using a 20× protein carrier in pMHC analyses and a high (~100×) and low (~9×) protein carrier in pTyr analyses, using CDK4/6 inhibitors and EGF stimulation to drive dynamic changes in the immunopeptidome and phosphoproteome, respectively. In both applications, inclusion of a protein carrier resulted in an increased number of MHC peptide or phosphopeptide identifications, as expected. At the same time, quantitative accuracy was adversely affected by the presence of the protein carrier, altering interpretation of the underlying biological response to perturbation. Moreover, for tyrosine phosphoproteomics, the presence of high levels of protein carrier led to a large number of missing values for endogenous phosphopeptides, leading to fewer quantifiable peptides relative to the “no-boost” condition. These data highlight the unique limitations and future experimental considerations for both analysis types and provide a framework for assessing quantitative accuracy in protein carrier experiments moving forward.

Mcl-1 is a highly labile protein, subject to extensive post-translational regulation. This distinguishes Mcl-1 from other antiapoptotic proteins and necessitates further study to better understand how interactions with proapoptotic Bcl-2 proteins affect its regulation. One such protein, Bim, is known to stabilize Mcl-1, and Bim phosphorylation has been associated with increased Mcl-1 binding. Consequently, we investigated the potential impact of Bim phosphorylation on Mcl-1 stability. We found that Bim stabilizes and primes Mcl-1 in RPCI-WM1 cells and is constitutively phosphorylated. Additionally, introduction of several phospho-mimetic and unphosphosphorylateable Bim mutations resulted in altered Mcl-1 stability and distinct Bim binding to antiapoptotic proteins. These findings suggest Bim phosphorylation not only regulates Mcl-1 stability but also is a potential mechanism for enforcing Mcl-1 dependence.

Utilizing a protein carrier in combination with isobaric labeling to "boost" the signal of other low-level samples in multiplexed analyses has emerged as an attractive strategy to enhance data quantity while minimizing protein input in mass spectrometry analyses. Recent applications of this approach include pMHC profiling and tyrosine phosphoproteomics, two applications that are often limited by large sample requirements. While including a protein carrier has been shown to increase the number of identifiable peptides in both applications, the impact of a protein carrier on quantitative accuracy remains to be thoroughly explored, particularly in relevant biological contexts where samples exhibit dynamic changes in abundance across peptides. Here, we describe two sets of analyses comparing MS2-based quantitation using a 20x protein carrier in pMHC analyses and a high (~100x) and low (~9x) protein carrier in pTyr analyses, using CDK4/6 inhibitors and EGF stimulation to drive dynamic changes in the immunopeptidome and phosphoproteome, respectively. In both applications, inclusion of a protein carrier resulted in an increased number of MHC peptide or phosphopeptide identifications, as expected. At the same time, quantitative accuracy was adversely affected by the presence of the protein carrier, altering interpretation of the underlying biological response to perturbation. Moreover, for tyrosine phosphoproteomics, the presence of high levels of protein carrier led to a large number of missing values for endogenous phosphopeptides, leading to fewer quantifiable peptides relative to the control condition. These data highlight the unique limitations and future experimental considerations for both analysis types and provide a framework for assessing quantitative accuracy in protein carrier experiments moving forward.

While direct inhibition of Bcl-2 and/or Bcl-xL is an exciting new approach in the treatment of hematologic malignancies, current agents in clinical testing including navitoclax (ABT-263) and ABT-199 are not predicted to be efficacious in most cases of multiple myeloma. Navitoclax and the related molecule ABT-737 promote apoptosis by releasing the pro-apoptotic BH3 only protein Bim from Bcl-2 and Bcl-xL, but are incapable of disrupting the interaction between Bim and Mcl-1, the predominant anti-apoptotic protein in both normal and malignant plasma cells. However, despite their dependence on Mcl-1, some human myeloma cell lines (HMCL) as well as freshly isolated patient-derived myeloma cells are sensitive to ABT-737 in vitro. Knowing that myeloma normally requires the bone marrow niche for survival, we hypothesized that a stromal derived factor might mediate the resistance to ABT-737 in vivo and identified IL-6 as a key resistance factor. In our initial experiments, the HS-5 stromal cell line induced resistance to ABT-737 in the HMCL MM.1s, as did conditioned media from both HS-5 cells and patient derived bone marrow stromal cells. Blocking IL-6 with an IL-6 neutralizing antibody reversed the protective effect of conditioned media, while addition of 10 ng/ml IL-6 protected cells to the same degree as conditioned media. In order to understand the mechanisms of IL-6 mediated resistance to ABT-737, we have focused on the effect of IL-6 on the Bcl-2 family of proteins. In previous work from our lab, the ABT-737 sensitive HMCLs KMS18, MM.1s, and 8226 all showed increased binding of Bim to Bcl-2 and Bcl-xL compared to ABT-737 resistant lines. We therefore examined the effect of IL-6 on the distribution of Bim among Bcl-2, Bcl-xL, and Mcl-1. In KMS18, stimulation with 10 ng/ml IL-6 for 24 hours increases binding of Bim to Mcl-1. The increased binding correlates with a 2 fold increased Mcl-1 expression at both the RNA and protein level. The increased Mcl-1 expression in response to IL-6 may be limited to KMS18 as it was not observed in MM.1s, 8226, or KMS11. IL-6 also does not prevent ABT-737 from disrupting the interaction between Bim and Bcl-xL. We also examined Bim for IL-6 induced post-translational modifications that could alter its binding to Bcl-2 proteins. Bim is known to be phosphorylated on serine 69 by Erk in response to growth factor stimulation. We observed Bim serine 69 phosphorylation within 5 minutes of IL-6 stimulation in both KMS18 and MM.1s cells. Phosphorylation was reversible with 10 μM of the MEK inhibitor U0126. Although serine 69 phosphorylation has been reported to result in Bim degradation, we do not observe any change in Bim levels over the course of 24 hours. Interestingly, the MEK inhibitor sensitized both KMS18 and MM.1s to ABT-737 and was able to partially overcome IL-6 induced resistance. Inhibition of Akt with the PI3K inhibitor LY294002 had no effect on Bim serine 69 phosphorylation or IL-6 induced resistance. Of note, the MEK inhibitor failed to prevent upregulation of Mcl-1 in KMS18, suggesting an alternative pathway mediates this effect. We are currently studying the role of JAK signaling in Mcl-1 expression with the inhibitors AZD1480 and ruxolitinib, and are also extending our results to additional cell lines and patient samples. These results suggest that targeting IL-6 or its downstream pathways may sensitize myeloma to Bcl-2 antagonists such as ABT-199 and navitoclax. Disclosures: Kaufman: Onyx: Consultancy; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Millennium Pharmaceuticals: Consultancy; Jansenn: Consultancy; Merck: Research Funding. Lonial:Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy. Boise:Onyx Pharmaceuticals: Consultancy.

Multiple myeloma cells are strongly dependent on the anti-apoptotic protein Mcl-1 for survival. However, several studies have shown that some myeloma cells are sensitive to treatment with ABT-737 and related compounds, which target Bcl-2 and Bcl-xL, but not Mcl-1. These studies suggest that sensitive cells have more of the direct-activating, pro-apoptotic protein Bim bound to Bcl-2 and Bcl-xL than to Mcl-1. We have previously shown that the expression levels of pro- and anti-apoptotic proteins have little influence on which anti-apoptotic protein binds Bim, leading us to further explore what factors influence the preferential binding of Bim. We have found that IL-6 stimulation of myeloma cells results in both the phosphorylation of Bim at serine 69, and a shift in Bim from Bcl-2 and Bcl-xL to Mcl-1. These observations suggest a role for phosphorylation in determining the anti-apoptotic protein that Bim binds to. To determine if constitutive phosphorylation of Bim regulates the steady state binding of Bim to anti-apoptotic Bcl-2 family members, we utilized phospho-affinity gel electrophoresis (Phos-Tag). We determined that Bim is constitutively phosphorylated in unstimulated myeloma cells. However, unlike what is observed following IL-6 stimulation, phosphorylation does not occur at serine 69. Through use of co-immunopreciptation studies, we found that phosphorylation status of Bim affects its binding to anti-apoptotic proteins. The phosphorylated species are preferentially bound to Bcl-2 and Bcl-xL, and not to Mcl-1. Additionally, we observed that phosphorylated Bim species bound to Bcl-2 and Bcl-xL were present at a significantly lower level in myeloma cells that were more dependent on Mcl-1, such as OPM2, than in KMS18 cells, which exhibited greater Bcl-2/Bcl-xL dependency. This pattern also held true for myeloma cells that had been selected for resistance to ABT-737. ABT-737 resistant KMS18 cells had no detectable phosphorylated species of Bim, and all of the Bim was bound to Mcl-1. We have begun to identify the phosphorylated amino acids in Bim through the use of phospho-Bim-specific antibodies. In addition to serine 69, we have determined that the constitutive phosphorylation does not occur at serine 59 or serine 87. However, preliminary data suggests that Bim phosphorylated at threonine 116 preferentially binds to Bcl-xL in KMS18 cells. We have also taken initial steps to identify signaling pathways and kinases that are phosphorylating Bim, and altering its affinity. We treated KMS18 myeloma cells for 12 hours with 10 μM of the MEK inhibitor U0126, the JNK inhibitor SP600125, or the p38 inhibitor SB203580, and ran lysates from each of these conditions on PhosTag gels. While U0126 and SB203580 treatment did not result in the loss of any phosphorylated Bim species, treatment with SP600125 resulted in a loss of phosphorylated Bim species comparable to treatment of myeloma cell lysates with lambda protein phosphatase. These results suggest that JNK mediates phosphorylation of Bim in myeloma cells. Interestingly, threonine 116 is contained within a JNK consensus sequence. Therefore, JNK could directly influence the preferential binding of Bim. Together, these results suggest that combining Bim kinase inhibitors with agents that result in decreased Mcl-1 (e.g. CDK9 inhibitors) or inducers of the Mcl-1 inhibitor Noxa (proteasome inhibitors) represents a novel approach in cancer cells that demonstrate Mcl-1 dependence. Disclosures: Boise: Onyx Pharmaceuticals: Consultancy.