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HACS1 is a Src homology 3 and sterile alpha motif domain-containing adaptor that is preferentially expressed in normal hematopoietic tissues and malignancies including myeloid leukemia, lymphoma, and myeloma. Microarray data showed HACS1 expression is up-regulated in activated human B cells treated with interleukin (IL)-4, CD40L, and anti-immunoglobulin (Ig)M and clustered with genes involved in signaling, including TNF receptor-associated protein 1 , signaling lymphocytic activation molecule , IL-6 , and DEC205 . Immunoblot analysis demonstrated that HACS1 is up-regulated by IL-4, IL-13, anti-IgM, and anti-CD40 in human peripheral blood B cells. In murine spleen B cells, Hacs1 can also be up-regulated by lipopolysaccharide but not IL-13. Induction of Hacs1 by IL-4 is dependent on Stat6 signaling and can also be impaired by inhibitors of phosphatidylinositol 3-kinase, protein kinase C, and nuclear factor B. HACS1 associates with tyrosine-phosphorylated proteins after B cell activation and binds in vitro to the inhibitory molecule paired Ig-like receptor B. Overexpression of HACS1 in murine spleen B cells resulted in a down-regulation of the activation marker CD23 and enhancement of CD138 expression, IgM secretion, and Xbp-1 expression. Knock down of HACS1 in a human B lymphoma cell line by small interfering ribonucleic acid did not significantly change IL-4-stimulated B cell proliferation. Our study demonstrates that HACS1 is up-regulated by B cell activation signals and is a participant in B cell activation and differentiation.

Purpose Lenalidomide is an oral immunomodulatory drug with multiple effects on the immune system and tumor cell microenvironment leading to inhibition of malignant cell growth. Based on encouraging reports of lenalidomide in relapsed and refractory chronic lymphocytic leukemia (CLL), we investigated the first-line use of single-agent lenalidomide in CLL. Patients and Methods Using a starting dose of lenalidomide 10 mg/d for 21 days of a 28-day cycle and weekly 5-mg dose escalations to a target of 25 mg, we encountered severe toxicities (tumor lysis, fatal sepsis) in the first two patients enrolled. The study was halted and the protocol amended to a more conservative regimen: starting dose of lenalidomide 2.5 mg with monthly escalations to a target dose of 10 mg, and extended tumor lysis prophylaxis and monitoring. Gene expression profiles from patient samples before and after 7 days of lenalidomide were performed. Results Twenty-five patients were enrolled on the amended protocol. No further tumor lysis events were reported. Tumor flare was common (88%) but mild. Grade 3 to 4 neutropenia occurred in 72% of patients, with only five episodes of febrile neutropenia. The overall response rate was 56% (no complete responses). Although rapid peripheral lymphocyte reductions were observed, rebound lymphocytoses during the week off-therapy were common. Lenalidomide-induced molecular changes enriched for cytoskeletal and immune-related genes were identified. Conclusion Lenalidomide is clinically active as first-line CLL therapy and is well-tolerated if a conservative approach with slow dose escalation is used. A lenalidomide-induced molecular signature provides insights into its immunomodulatory mechanisms of action in CLL.

The association of fibroblast growth factor receptor 3 (FGFR3) expression with t(4;14) multiple myeloma (MM) and the demonstration of the transforming potential of this receptor tyrosine kinase (RTK) make it a particularly attractive target for drug development. We report here a novel and highly specific anti-FGFR3-neutralizing antibody (PRO-001). PRO IntroductionTreatment of multiple myeloma (MM) has advanced over the past decade, resulting in prolongation of median survival from 3 to 5 years. Despite the improved outcome with treatment regimens that include dose intensification, patients invariably relapse, and MM remains a universally fatal disease. [1][2][3] Because the limits of current chemotherapy have been reached, new approaches to therapy are urgently required. Various studies have delineated fundamental genetic lesions in MM that affect well-defined oncogenic pathways, growth, and survival signaling cascades. 4,5 These key cellular and genetic pathogenic processes provide a framework to identify novel therapeutic targets.The t(4;14)(p16.3;q32) translocation, which occurs in approximately 15% to 20% of MM tumors, 6,7 results in the dysregulated expression of 2 putative oncogenes, MMSET and fibroblast growth factor receptor 3 (FGFR3). 8 FGFR3 belongs to a family of 5 receptors, FGFR1-5, 4 of which harbor a functional tyrosine kinase. The FGFRs are characterized by 2 to 3 immunoglobulin (Ig)-like extracellular domains that bind ligand, a hydrophobic transmembrane domain, and a cytoplasmic region that contains a split tyrosine kinase domain. 9 Binding of fibroblast growth factor (FGF) ligand and heparin promotes receptor dimerization and activation of the kinase domain, resulting in autophosphorylation of specific tyrosines. Activation of FGFRs transduces signals through mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K) pathways, among others that regulate multiple cellular processes, including cell growth, differentiation, migration, and survival depending on the cellular context. 9,10 Studies indicate that FGFR3 may play a significant, albeit not a singular, role in myeloma oncogenesis, thus making this receptor tyrosine kinase (RTK) an attractive target for therapeutic intervention. Activation of wild-type (WT) FGFR3 promotes proliferation of myeloma cells and is weakly transforming in a hematopoietic mouse model. 11,12 Subsequent acquisition of activating mutations of FGFR3 in some MMs is associated with disease progression and is strongly transforming in several experimental models. 12,13 In vitro studies suggest that FGFR3 can impart chemoresistance 14 consistent with clinical data that demonstrate poor responses to conventional chemotherapy 15,16 and shortened median survival of t(4;14) MM patients. 15,17,18 The data have spurred the development of selective FGFR3 tyrosine kinase inhibitors for the potential treatment of MM. To date, several small-molecule inhibitors have been reported to induce cytotoxic responses of FGFR3-expressing myeloma cells. [19][20][21][22...

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Activating mutations of the MAPK pathway are reported in over half of myeloma tumors. Experience with MEK inhibitors in solid tumors suggest that although tumors harboring BRAF or RAS mutations are more likely to respond, response rates are low and duration of responses short. Potential explanations include the activation of alternative signaling pathways and in particular PI3K/AKT signaling. Studies of multiple myeloma (MM) tumors suggest that AKT activation is independent of oncogenic RAS and that combined inhibition of RAS and AKT enhances MM cell death. Based on these observations, we initiated a clinical trial to evaluate the activity of trametinib (TMTB) in MM patients (pts) with or without RAS/RAF mutations, as a single-agent and in combination with AKT inhibition for pts who fail to respond to TMTB alone. Methods: Pts were independently recruited into biomarker positive (K/NRAS or BRAF mutated) or biomarker negative (K/NRAS, BRAF wild type) groups. All pts received TMTB, 2 mg/day on a 28 day cycle. In pts who developed progressive disease (PD) or achieved less than a partial response (PR) after 4 cycles of TMTB monotherapy, GSK2141795 (pan-AKT inhibitor) was added. TMTB combined with GSK2141795 was dosed at 1.5 mg/50 mg taken daily. The M-protein at the time of adding GSK2141795 was considered the new baseline for response assessment to the combination. The main objectives were to evaluate overall response rates (ORR; IMWG) to TMTB in the 2 groups and determine the clinical benefit of adding GSK2141795 to TMTB. Results: At the data cutoff, 25 pts were enrolled: 12 in the mutated (MUT) and 13 in the wild type (WT) groups. Median age and prior lines of therapy were 65 years (range 42-76) and 4 (range 4-8) for the MUT group and 64 years (range 51-81) and 4 (range 2-8) for the WT group. 96%, 100%, 60% and 96% of pts had received prior proteasome inhibitor (PI), immunomodulatory drugs (IMiDs), pomalidomide or PI+IMiD therapy, respectively. The most common (>30%) adverse events (AEs) possibly related to study drugs were thrombocytopenia (40%) and diarrhea (32%) for TMTB monotherapy and nausea (72%), rash (63%), thrombocytopenia (36%) and anorexia (36%) with the addition of GSK2141795. The most frequent grade 3-4 AEs (>20%) were thrombocytopenia (24%) for TMTB monotherapy and thrombocytopenia (45%), anemia (45%), lymphopenia (36%), hyponatremia (27%), and neutropenia (27%) in combination with GSK2141795. One death occurred due to gastrointestinal bleed unrelated to study drugs. Of 24 pts assessable for response, confirmed ORR was 8% (1 PR) for the MUT group (N=12), Further, 1 pt had an unconfirmed minimal response (MR) and 4 pts had stable disease (SD). For the WT group (N=12), we observed 2 MR (1unconfirmed) and 3 SD. For clinical benefit rate (CBR) of 17% in both groups. With the addition of GSK2141795 to TMTB (N=11), the ORR was 27% (3 PR, 1 unconfirmed) and 1 pt had an MR (CBR=36%). The median PFS is estimated to be 3.2 (range 0.9-3.8) and 1.8 (range 0.9-NR) months (p=0.91), for the MUT and WT groups, respectively. Correlative studies are ongoing and are designed to identify predictors of response and resistance to TMTB. These include, assessment of pre- and post-treatment expression of phospho-ERK1/2, serial monitoring of clonal dynamics in bone marrow and cell-free DNA and pharmacogenomic studies. Analyses will be presented. Conclusions: The MEK1/2 inhibitor, TMTB demonstrates clinical activity in MM pts with RAS mutated tumors. In addition, disease control was observed for pts with WT RAS/RAF tumors. However, single agent activity in heavily pre-treated pts is modest. The addition of GSK2141795 to block the alternative signaling pathway improved the ORR to 27% supporting further exploration of this treatment strategy for MM. Disclosures Trudel: Celgene: Consultancy, Equity Ownership, Honoraria; Novartis: Consultancy, Honoraria; Glaxo Smith Kline: Honoraria, Research Funding; Oncoethix: Research Funding; BMS: Honoraria; Amgen: Honoraria. Bahlis:Amgen: Consultancy, Honoraria; BMS: Honoraria; Janssen: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau; Onyx: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau. Venner:Amgen: Honoraria; Takeda: Honoraria; Celgene: Honoraria, Research Funding; J+J: Research Funding; Janssen: Honoraria. Hay:Amgen: Research Funding; Novartis: Research Funding; Janssen: Research Funding; Kite Pharmaceuticals: Research Funding.

Molecular programs that underlie precursor progression in multiple myeloma are incompletely understood. Here, we report a disease spectrum-spanning, single-cell analysis of the Vκ*MYC myeloma mouse model. Using samples obtained from mice with serologically undetectable disease, we identify malignant cells as early as 30 weeks of age and show that these tumours contain subclonal copy number variations that persist throughout progression. We detect intratumoural heterogeneity driven by transcriptional variability during active disease and show that subclonal expression programs are enriched at different times throughout early disease. We then show how one subclonal program related to GCN2 stress response is progressively activated during progression in myeloma patients. Finally, we use chemical and genetic perturbation of GCN2 in vitro to support this pathway as a therapeutic target in myeloma. These findings therefore present a model of precursor progression in Vκ*MYC mice, nominate an adaptive mechanism important for myeloma survival, and highlight the need for single-cell analyses to understand the biological underpinnings of disease progression.

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