Justin King

3D tissue-engineered bone marrow as a novel model to study pathophysiology and drug resistance in multiple myeloma.

PURPOSE Multiple myeloma (MM) is the second most prevalent hematological malignancy and it remains incurable despite the introduction of several novel drugs. The discrepancy between preclinical and clinical outcomes can be attributed to the failure of classic two-dimensional (2D) culture models to accurately recapitulate the complex biology of MM and drug responses observed in patients. EXPERIMENTAL DESIGN We developed 3D tissue engineered bone marrow (3DTEBM) cultures derived from the BM supernatant of MM patients to incorporate different BM components including MM cells, stromal cells, and endothelial cells. Distribution and growth were analyzed by confocal imaging, and cell proliferation of cell lines and primary MM cells was tested by flow cytometry. Oxygen and drug gradients were evaluated by immunohistochemistry and flow cytometry, and drug resistance was studied by flow cytometry. RESULTS 3DTEBM cultures allowed proliferation of MM cells, recapitulated their interaction with the microenvironment, recreated 3D aspects observed in the bone marrow niche (such as oxygen and drug gradients), and induced drug resistance in MM cells more than 2D or commercial 3D tissue culture systems. CONCLUSIONS 3DTEBM cultures not only provide a better model for investigating the pathophysiology of MM, but also serve as a tool for drug development and screening in MM. In the future, we will use the 3DTEBM cultures for developing personalized therapeutic strategies for individual MM patients.

CD56bright NK cells exhibit potent antitumor responses following IL-15 priming

NK cells, lymphocytes of the innate immune system, are important for defense against infectious pathogens and cancer. Classically, the CD56dim NK cell subset is thought to mediate antitumor responses, whereas the CD56bright subset is involved in immunomodulation. Here, we challenge this paradigm by demonstrating that brief priming with IL-15 markedly enhanced the antitumor response of CD56bright NK cells. Priming improved multiple CD56bright cell functions: degranulation, cytotoxicity, and cytokine production. Primed CD56bright cells from leukemia patients demonstrated enhanced responses to autologous blasts in vitro, and primed CD56bright cells controlled leukemia cells in vivo in a murine xenograft model. Primed CD56bright cells from multiple myeloma (MM) patients displayed superior responses to autologous myeloma targets, and furthermore, CD56bright NK cells from MM patients primed with the IL-15 receptor agonist ALT-803 in vivo displayed enhanced ex vivo functional responses to MM targets. Effector mechanisms contributing to IL-15–based priming included improved cytotoxic protein expression, target cell conjugation, and LFA-1–, CD2-, and NKG2D-dependent activation of NK cells. Finally, IL-15 robustly stimulated the PI3K/Akt/mTOR and MEK/ERK pathways in CD56bright compared with CD56dim NK cells, and blockade of these pathways attenuated antitumor responses. These findings identify CD56bright NK cells as potent antitumor effectors that warrant further investigation as a cancer immunotherapy.

A CD138-independent strategy to detect minimal residual disease and circulating tumour cells in multiple myeloma.

CD138 (also termed SDC1) has been the gold‐standard surface marker to detect multiple myeloma (MM) cells for decades; however, drug‐resistant residual and circulating MM cells were shown to have lower expression of this marker. In this study, we have shown that residual MM cells following bortezomib treatment are hypoxic. This combination of drug exposure and hypoxia down‐regulates their CD138 expression, thereby making this marker unsuitable for detecting residual or other hypoxic MM cells, such as circulating tumour cells, in MM. Hence, we developed an alternative biomarker set which detects myeloma cells independent of their hypoxic and CD138 expression status in vitro, in vivo and in primary MM patients. The new markers were able to identify a clonal CD138‐negative population as minimal residual disease in the bone marrow and peripheral blood of MM patients. Further investigation to characterize the role of this population as a prognostic marker in MM is warranted.

Personalization of cancer treatment using predictive simulation

BackgroundThe personalization of cancer treatments implies the reconsideration of a one-size-fits-all paradigm. This move has spawned increased use of next generation sequencing to understand mutations and copy number aberrations in cancer cells. Initial personalization successes have been primarily driven by drugs targeting one patient-specific oncogene (e.g., Gleevec, Xalkori, Herceptin). Unfortunately, most cancers include a multitude of aberrations, and the overall impact on cancer signaling and metabolic networks cannot be easily nullified by a single drug.MethodsWe used a novel predictive simulation approach to create an avatar of patient cancer cells using point mutations and copy number aberration data. Simulation avatars of myeloma patients were functionally screened using various molecularly targeted drugs both individually and in combination to identify drugs that are efficacious and synergistic. Repurposing of drugs that are FDA-approved or under clinical study with validated clinical safety and pharmacokinetic data can provide a rapid translational path to the clinic. High-risk multiple myeloma patients were modeled, and the simulation predictions were assessed ex vivo using patient cells.ResultsHere, we present an approach to address the key challenge of interpreting patient profiling genomic signatures into actionable clinical insights to make the personalization of cancer therapy a practical reality. Through the rational design of personalized treatments, our approach also targets multiple patient-relevant pathways to address the emergence of single therapy resistance. Our predictive platform identified drug regimens for four high-risk multiple myeloma patients. The predicted regimes were found to be effective in ex vivo analyses using patient cells.ConclusionsThese multiple validations confirm this approach and methodology for the use of big data to create personalized therapeutics using predictive simulation approaches.

Enhancing proteasome-inhibitory activity and specificity of bortezomib by CD38 targeted nanoparticles in multiple myeloma

Abstract The establishment of more effective treatments that can circumvent chemoresistance in Multiple Myeloma (MM) is a priority. Although bortezomib (BTZ) is one of the most potent proteasome inhibitors available, still possesses limitations related to dose limiting side effects. Several strategies have been developed to improve the delivery of chemotherapies to MM by targeting different moieties expressed on MM cells to nanoparticle delivery systems (NPs), which have failed mainly due to their heterogeneous expression on these cells. Our goal was to test CD38 targeted chitosan NPs as novel targeting moiety for MM to improve the potency and efficacy of BTZ in MM cells and reduce the side effects in healthy tissue. We have showed preferential BTZ release in tumor‐microenvironment, specific binding to MM cells, and an improved drug cellular uptake through BTZ diffusion from the surface and endocytosed NPs, which translated in enhanced proteasome inhibition and robust cytotoxic effect on MM cells when BTZ was administered through anti‐CD38 chitosan NPs. Furthermore, the anti‐CD38 chitosan NPs specifically delivered therapeutic agents to MM cells improving therapeutic efficacy and reducing side effects in vivo. The anti‐CD38 chitosan NPs showed low toxicity profile allowing enhancement of proteasome‐inhibitory activity and specificity of BTZ by endocytosis‐mediated uptake of CD38 representing a promising therapy in MM. Graphical abstract Figure. No Caption available.

Newly established myeloma-derived stromal cell line MSP-1 supports Multiple Myeloma proliferation, migration, and adhesion and induces drug resistance more than normal-derived stroma

The effects of a normal bone marrow (BM) microenvironment compared to a malignant microenvironment on multiple myeloma (MM) are significantly different, where BM stromal cells (BMSCs) play a key role. We established, characterized and compared a myeloma-derived stromal cell line (Myeloma Stromal Puente-1, MSP-1) with two normal stromal cell lines (HS-5 and HS-27A). MSP-1 was found to affect MM proliferation, adhesion, migration and drug resistance in a more profound manner than HS-5 and HS-27A. These results demonstrated the importance of malignant versus normal BM microenvironment on several key MM processes, providing a new myeloma-derived stromal cell line to study the effect of tumor microenvironment on MM.

The efficacy of salvage autologous stem cell transplant among patients with multiple myeloma who received maintenance therapy post initial transplant

Autologous stem cell transplant (ASCT) remains a standard of care for patients with multiple myeloma (MM) even in the era of novel drugs. However, MM remains incurable and most patients relapse after ASCT. A second ASCT is one of the treatment options for these patients with relapsed MM that offers additional progression-free survival (PFS) and overall survival (OS) advantage over conventional chemotherapy [1, 2]. Studies conducted in an era before the routine use of maintenance therapy post-ASCT have shown PFS to be about 40–50% of what is achieved after a first ASCT [3]. However, there is a lack of data on survival outcomes after 2nd ASCT in the era of modern maintenance therapy. Here, we report outcomes of patients undergoing 2nd ASCT who received maintenance therapy after the initial ASCT in comparison with a cohort who did not receive maintenance. We retrospectively reviewed data for patients who underwent a 2nd stem cell transplant at Washington University in St. Louis from 2008 to 2016. Patients undergoing 2nd transplant as part of tandem ASCT or allogeneic or syngeneic transplants were excluded. We identified two cohorts of patients. One cohort included 30 patients who underwent 2nd ASCT for salvage after receiving maintenance with lenalidomide or bortezomib following initial ASCT. The other was a comparison cohort of 33 patients who underwent initial and 2nd ASCT without maintenance. Relevant clinical data was collected through chart abstraction. Disease staging was assessed as per International Staging System (ISS) for MM [4]. Cytogenetic abnormalities, identified by fluorescent in situ hybridization studies and conventional karyotype were classified as standard-risk, intermediate-risk, and high-risk cytogenetic abnormalities [5]. Response was assessed according to International Myeloma Working Group definitions [6]. PFS and OS were assessed by Kaplan–Meier curves. The primary endpoint was comparison of median PFS following 2nd ASCT in relation to PFS following initial ASCT (PFS2/ PFS1) between the two cohorts. All statistical analysis was performed using SPSS Ver 24 (IBM Corp). Demographic data for the two cohorts are summarized in Table 1. The two cohorts were similar in respect to demographics and staging. Overall the median age at 2nd ASCT was 62 years (range 44–74 years), and 52% of patients were male. Transplant characteristics and outcomes are detailed in Table 2. Prior to initial ASCT, the majority of patients received induction with proteasome inhibitor (PI) or immunomodulatory drugs (IMID)-based regimens, or combination PI+ IMID. All patients underwent their 1st ASCT with melphalan conditioning. Overall the complete response (CR) rate was 44%, 40% in the maintenance cohort and 48% in the no maintenance cohort. Following initial ASCT, 28 patients received maintenance lenalidomide and 2 received bortezomib due to adverse effects related to lenalidomide. Median PFS after the initial ASCT for these patients was 31.3 months (range 17.2–56.7 months). The median PFS after initial ASCT for the 33 patients not receiving maintenance was 37.1 months (range 17.7–93.6). Upon relapse after the initial ASCT, the majority of patients received re-induction with PI or IMID-based, or PI + IMID regimens, while 11% received no re-induction. Melphalan alone was used as the conditioning regimen in * Ravi Vij rvij@wustl.edu

A multiple myeloma-specific capture sequencing platform discovers novel translocations and frequent, risk-associated point mutations in IGLL5

Multiple myeloma (MM) is a disease of copy number variants (CNVs), chromosomal translocations, and single-nucleotide variants (SNVs). To enable integrative studies across these diverse mutation types, we developed a capture-based sequencing platform to detect their occurrence in 465 genes altered in MM and used it to sequence 95 primary tumor-normal pairs to a mean depth of 104×. We detected cases of hyperdiploidy (23%), deletions of 1p (8%), 6q (21%), 8p (17%), 14q (16%), 16q (22%), and 17p (4%), and amplification of 1q (19%). We also detected IGH and MYC translocations near expected frequencies and non-silent SNVs in NRAS (24%), KRAS (21%), FAM46C (17%), TP53 (9%), DIS3 (9%), and BRAF (3%). We discovered frequent mutations in IGLL5 (18%) that were mutually exclusive of RAS mutations and associated with increased risk of disease progression (p = 0.03), suggesting that IGLL5 may be a stratifying biomarker. We identified novel IGLL5/IGH translocations in two samples. We subjected 15 of the pairs to ultra-deep sequencing (1259×) and found that although depth correlated with number of mutations detected (p = 0.001), depth past ~300× added little. The platform provides cost-effective genomic analysis for research and may be useful in individualizing treatment decisions in clinical settings.

Abstract 5305: Predicting relapse using CD138-independent strategy to detect residual myeloma plasma cells

Introduction: Despite the introduction of novel therapies, majority of multiple myeloma (MM) patients relapses. The major reason for the relapse in MM is the presence of a minimal residual disease (MRD) population that remains after treatment. MM cells are defined as CD138+; however, the presence of clonogenic CD138-negative MM cells and hypoxia-driven CD138 decrease in expression were previously shown. We propose a novel set of biomarkers (independent of CD138) to detect MRD in MM in order to predict patient relapse. Procedures: We tested the effect of hypoxia on CD markers expression in MM cell lines. We developed new method to detect MM cells by flow cytometry based on CD38(APC+) gating and negatively selecting other leukocytes including T cells, B cells, monocytes, NK cells, and dendritic cells using CD3, CD19, CD14, CD16, and CD123, respectively (FITC-conjugated), the MM cells were defined as APC+/FITC-. We compared traditional method (CD138-based) with the new set of biomarkers (1) to detect normoxic and hypoxic MM cells, (2) to detect MM cells in the peripheral blood (PB) and the bone marrow (BM) from MM patients, confirming cell clonality by internal staining with Kappa/Lambda light chain antibodies, and (3) to predict relapse in MM patients whose BM was defined a CD138-negative. Summary of data: We found that hypoxia decreased the expression of CD138 by ∼50%, while CD38 was not altered by hypoxia in MM cells. The use of CD138+ as a marker for MM detected only 60-75% of hypoxic MM cells, whereas APC+/FITC- strategy detected close to a 100% of the hypoxic MM cells in vitro. In patient samples, all patients had a higher number of BM-derived and circulating MM cells as detected by APC+/FITC- strategy compared to CD138+. In addition, the clonality of the APC+/FITC- population in BM negative fractions was the same as the original disease (CD138+ cells). Moreover, studies conducted on 16 patients with BM defined as a CD138-negative, showed that the novel APC+/FITC- strategy was more successful than CD138-based method or histology in predicting recurrence and time to relapse. The median time-to-progression with Conclusion: CD138 cannot be used as a universal marker to detect MM cells, especially when they are hypoxic. A novel set of biomarkers CD38+/CD3-/CD19-/CD14-/CD16-/CD123- (APC+/FITC-) was designed to detect MM cells independent of CD138 expression and hypoxic status. This strategy was able to detect a clonal CD138-negative MM cell population in the BM and PB from MM patients. Moreover, this strategy predicted tumor recurrence and relapse in MM patients more accurately compared to traditional methods such as CD138-based or histology. Citation Format: Barbara Muz, Feda Azab, Pilar de la Puente, Justin King, Micah Luderer, Ravi Vij, Abdel Kareem Azab. Predicting relapse using CD138-independent strategy to detect residual myeloma plasma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5305. doi:10.1158/1538-7445.AM2015-5305

Abstract 5356: 3D tissue-engineered bone marrow niche as novel method to study pathophysiology and drug resistance in multiple myeloma

INTRODUCTION: Current methods to assess multiple myeloma (MM) cancer treatments are often inaccurate, in part, due to these methods cannot take into consideration the variability of every patient, and fail to recreate the bone marrow (BM) microenvironment. In this study we developed a patient-derived three-dimensional tissue-engineered BM (3DTEBM) niche as novel model that will allow better evaluation of interactions of MM cells and their microenvironment and how these interactions may affect MM drug resistance in the specific patient. METHODS & RESULTS: 3DTEBM cultures were made by crosslinking of patient-derived BM supernatants; the 3D-scaffold included MM cell lines or primary CD138+ MM cells and other accessory cells (stromal cells and endothelial cells, or CD138- cells). 3DTEBM cultures were studied for cell proliferation by flow cytometry, and compared with 2D cultures and other commercial 3D systems including PLGA microspheres, Algimatrix, and Matrigel, and imaged for 2 weeks using a confocal microscope. 3DTEBM cultures promoted the proliferation of MM cells more than 2D cultures and commercial 3D systems. 3DTEBM cultures supported the growth of fresh and frozen primary MM cells and allowed the use of these cells ex vivo at least 2 weeks. In addition, we found a natural development of polarization in the cellularity of the scaffold, with the bottom rich of stromal cells, top rich of endothelial cells, and MM cells through all the parts of the scaffold, reflecting the pathophysiological development of MM in the BM niche. The effect of 3DTEBM cultures on cytokine expression was determined by cytokine antibody arrays and 3D-tissue depth effects in drug gradients, oxygenation levels and proliferation (HIF-1α, PIM, Ki67, CXCR4, and CD138 expression) were determined using flow cytometry or IHC. 3DTEBM cultures showed that 23 out of 80 cytokines tested were highly expressed with at least 3-fold increase in the 3D cultures vs 2D cultures. Furthermore, 3DTEBM cultures recreated the BM niche drug gradients and oxygenation levels due to depth of tissue; MM cells showed lower drug uptake and higher hypoxia levels (PIM and HIF-1α) with depth of tissue, and hypoxia-associated decreased CD138 and increased CXCR4 expression. We also found that MM cells were quiescent (lower Ki67 expression) in the hypoxic niche. Finally, drug resistance of MM cell lines or primary MM cells in 3DTEBM cultures was analyzed by flow cytometry. 3DTEBM cultures induced drug resistance in MM cells more than 2D cultures and commercial 3D systems, and preliminary results indicate that drug resistance profile in the 3DTEBM matched the clinical manifestation of the disease in the specific patient, while 2D cultures did not. CONCLUSION: The patient-derived 3DTEBM is a more biologically relevant model for evaluation of interactions of MM cells and their microenvironment in the BM niche, especially in the context of drug resistance. Citation Format: Pilar de la Puente, Rebecca Gilson, Barbara Muz, Feda Azab, Justin King, Samuel Achilefu, Ravi Vij, Abdel Kareem Azab. 3D tissue-engineered bone marrow niche as novel method to study pathophysiology and drug resistance in multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5356. doi:10.1158/1538-7445.AM2015-5356