Abstract
With the introduction of novel therapeutic agents, survival in Multiple Myeloma (MM) has increased in recent years. However, drug-resistant clones inevitably arise and lead to disease ...progression and death. The current International Myeloma Working Group response criteria are broad and make it difficult to clearly designate resistant and responsive patients thereby hampering proteo-genomic analysis for informative biomarkers for sensitivity. In this proof-of-concept study we addressed these challenges by combining an ex-vivo drug sensitivity testing platform with state-of-the-art proteomics analysis. 35 CD138-purified MM samples were taken from patients with newly diagnosed or relapsed MM and exposed to therapeutic agents from five therapeutic drug classes including Bortezomib, Quizinostat, Lenalidomide, Navitoclax and PF-04691502. Comparative proteomic analysis using liquid chromatography-mass spectrometry objectively determined the most and least sensitive patient groups. Using this approach several proteins of biological significance were identified in each drug class. In three of the five classes focal adhesion-related proteins predicted low sensitivity, suggesting that targeting this pathway could modulate cell adhesion mediated drug resistance. Using Receiver Operating Characteristic curve analysis, strong predictive power for the specificity and sensitivity of these potential biomarkers was identified. This approach has the potential to yield predictive theranostic protein panels that can inform therapeutic decision making.
Acute myeloid leukemia (AML) with co-occurring NUP98-NSD1 and FLT3-ITD is associated with unfavorable prognosis and represents a particularly challenging treatment group. To identify novel effective ...therapies for this AML subtype, we screened patient cells and engineered cell models with over 300 compounds. We found that mouse hematopoietic progenitors co-expressing NUP98-NSD1 and FLT3-ITD had significantly increased sensitivity to FLT3 and MEK-inhibitors compared to cells expressing either aberration alone (P < 0.001). The cells expressing NUP98-NSD1 alone had significantly increased sensitivity to BCL2-inhibitors (P = 0.029). Furthermore, NUP98-NSD1
/FLT3-ITD
patient cells were also very sensitive to BCL2-inhibitor navitoclax, although the highest select sensitivity was found to SRC/ABL-inhibitor dasatinib (mean IC
2.2 nM). Topoisomerase inhibitor mitoxantrone was the least effective drug against NUP98-NSD1
/FLT3-ITD
AML cells. Of the 25 significant hits, four remained significant also compared to NUP98-NSD1
/FLT3-ITD
AML patients. We found that SRC/ABL-inhibitor dasatinib is highly synergistic with BCL2-inhibitor navitoclax in NUP98-NSD1
/FLT3-ITD
cells. Gene expression analysis supported the potential relevance of dasatinib and navitoclax by revealing significantly higher expression of BCL2A1, FGR, and LCK in NUP98-NSD1
/FLT3-ITD
patients compared to healthy CD34+ cells. Our data suggest that dasatinib-navitoclax combination may offer a clinically relevant treatment strategy for AML with NUP98-NSD1 and concomitant FLT3-ITD.
Innate drug sensitivity in healthy cells aids identification of lineage specific anti-cancer therapies and reveals off-target effects. To characterize the diversity in drug responses in the major ...hematopoietic cell types, we simultaneously assessed their sensitivity to 71 small molecules utilizing a multi-parametric flow cytometry assay and mapped their proteomic and basal signaling profiles. Unsupervised hierarchical clustering identified distinct drug responses in healthy cell subsets based on their cellular lineage. Compared to other cell types, CD19
/B and CD56
/NK cells were more sensitive to dexamethasone, venetoclax and midostaurin, while monocytes were more sensitive to trametinib. Venetoclax exhibited dose-dependent cell selectivity that inversely correlated to STAT3 phosphorylation. Lineage specific effect of midostaurin was similarly detected in CD19
/B cells from healthy, acute myeloid leukemia and chronic lymphocytic leukemia samples. Comparison of drug responses in healthy and neoplastic cells showed that healthy cell responses are predictive of the corresponding malignant cell response. Taken together, understanding drug sensitivity in the healthy cell-of-origin provides opportunities to obtain a new level of therapy precision and avoid off-target toxicity.
The bone marrow (BM) provides a protective microenvironment to support the survival of leukemic cells and influence their response to therapeutic agents. In acute myeloid leukemia (AML), the high ...rate of relapse may in part be a result of the inability of current treatment to effectively overcome the protective influence of the BM niche. To better understand the effect of the BM microenvironment on drug responses in AML, we conducted a comprehensive evaluation of 304 inhibitors, including approved and investigational agents, comparing ex vivo responses of primary AML cells in BM stroma-derived and standard culture conditions. In the stroma-based conditions, the AML patient cells exhibited significantly reduced sensitivity to 12% of the tested compounds, including topoisomerase II, B-cell chronic lymphocytic leukemia/lymphoma 2 (BCL2), and many tyrosine kinase inhibitors (TKIs). The loss of TKI sensitivity was most pronounced in patient samples harboring FLT3 or PDGFRB alterations. In contrast, the stroma-derived conditions enhanced sensitivity to Janus kinase (JAK) inhibitors. Increased cell viability and resistance to specific drug classes in the BM stroma-derived conditions was a result of activation of alternative signaling pathways mediated by factors secreted by BM stromal cells and involved a switch from BCL2 to BCLXL-dependent cell survival. Moreover, the JAK1/2 inhibitor ruxolitinib restored sensitivity to the BCL2 inhibitor venetoclax in AML patient cells ex vivo in different model systems and in vivo in an AML xenograft mouse model. These findings highlight the potential of JAK inhibitors to counteract stroma-induced resistance to BCL2 inhibitors in AML.
•BM stroma-derived conditions protect AML patient cells against topoisomerase II and BCL2 inhibitors, as well as several classes of TKIs.•JAK1/2 inhibitor ruxolitinib reverses cytoprotection against BCL2 antagonist venetoclax, suggesting a novel combinatorial treatment.
Multiple myeloma (MM) is characterized by extensive immunoglobulin production leading to an excessive load on protein homeostasis in tumor cells. Aminopeptidases contribute to proteolysis by ...catalyzing the hydrolysis of amino acids from proteins or peptides and function downstream of the ubiquitin-proteasome pathway. Notably, aminopeptidases can be utilized in the delivery of antibody and peptide-conjugated drugs, such as melflufen, currently in clinical trials. We analyzed the expression of 39 aminopeptidase genes in MM samples from 122 patients treated at Finnish cancer centers and 892 patients from the CoMMpass database. Based on ranked abundance,
,
,
,
, and
were highly expressed in MM.
,
,
,
, and
were differentially expressed between relapsed/refractory and newly diagnosed MM samples (
< 0.05). Sensitivity to melflufen was detected ex vivo in 11/15 MM patient samples, and high sensitivity was observed, especially in relapsed/refractory samples. Survival analysis revealed that high expression of
,
,
, and
(
< 0.05) was associated with shorter overall survival. Hydrolysis analysis demonstrated that melflufen is a substrate for aminopeptidases LAP3, LTA4H, RNPEP, and ANPEP. The sensitivity of MM cell lines to melflufen was reduced by aminopeptidase inhibitors. These results indicate critical roles of aminopeptidases in disease progression and the activity of melflufen in MM.
Constitutive JAK/STAT3 signaling contributes to disease progression in many lymphoproliferative disorders. Recent genetic analyses have revealed gain-of-function
mutations in lymphoid cancers leading ...to hyperactivation of STAT3, which may represent a potential therapeutic target. Using a functional reporter assay, we screened 306 compounds with selective activity against various target molecules to identify drugs capable of inhibiting the cellular activity of STAT3. Top hits were further validated with additional models including STAT3-mutated natural killer (NK)-cell leukemia/lymphoma cell lines and primary large granular lymphocytic (LGL) leukemia cells to assess their ability to inhibit STAT3 phosphorylation and STAT3 dependent cell viability. We identified JAK, mTOR, Hsp90 and CDK inhibitors as potent inhibitors of both WT and mutant STAT3 activity. The Hsp90 inhibitor luminespib was highly effective at reducing the viability of mutant STAT3 NK cell lines and LGL leukemia patient samples. Luminespib decreased the phosphorylation of mutant STAT3 at Y705, whereas JAK1/JAK2 inhibitor ruxolitinib had reduced efficacy on mutant STAT3 phosphorylation. Additionally, combinations involving Hsp90, JAK and mTOR inhibitors were more effective at reducing cell viability than single agents. Our findings show alternative approaches to inhibit STAT3 activity and suggest Hsp90 as a therapeutic target in lymphoproliferative disorders with constitutively active STAT3.
Immunoglobulin light‐chain (AL) amyloidosis is a rare disease caused by clonal plasma cell secretion of misfolded light chains that assemble as toxic amyloid fibrils, depositing in vital organs ...including the heart and kidneys, causing organ dysfunction. Plasma cell–directed therapeutics are expected to reduce production of toxic light chain by eliminating amyloidogenic cells in bone marrow, thereby diminishing amyloid fibril deposition and providing the potential for organ recovery. Melphalan flufenamide (melflufen) is a first‐in‐class peptide–drug conjugate that targets aminopeptidases and rapidly releases alkylating agents inside tumor cells. Melflufen is highly lipophilic, permitting rapid uptake by cells, where it is enzymatically hydrolyzed by aminopeptidases, resulting in intracellular accumulation of the alkylating agents, including melphalan. Previous data demonstrating sensitivity of myeloma cells to melflufen suggest that the drug might be useful in AL amyloidosis. We describe the effects of melflufen on amyloidogenic plasma cells in vitro and ex vivo, demonstrating enhanced cytotoxic effects in comparison to melphalan, as well as novel mechanisms of action through the unfolded protein response (UPR) pathway. These findings provide evidence that melflufen‐mediated cytotoxicity extends to amyloidogenic plasma cells, and support the rationale for the evaluation of melflufen in patients with AL amyloidosis.
Introduction
Off-target cytotoxicity resulting in severe side effects and compromising patient survival often hampers the development of new cancer therapeutics. Understanding the complete drug ...response landscape of different cell populations is crucial to identify drugs that selectively eradicate the malignant cell population, but spare healthy cells. Here, we developed a high content, no wash, multi-parametric flow cytometry based assay that enables testing of blood cancer patient samples and simultaneously monitors the effects of several drugs on 11 hematopoietic cell types. The assay can be used to i) dissect malignant from healthy cell responses and predict off-target effects; ii) assess drug effects on immune cell subsets; iii) identify drugs that can potentially be repositioned to new blood cancer indications.
Methods
Mononuclear cells were prepared from bone marrow aspirates of 7 multiple myeloma (MM) and 3 acute myeloid leukemia (AML) patients plus the peripheral blood from a healthy donor, which were collected following informed consent and in compliance with the Declaration of Helsinki. Optimal cell density, antibody dilutions, incubation time, and wash versus no wash assay conditions for the selected antibody panels were determined. Cells were incubated at a density of 2 million cells/ml in either 96- or 384-well plates for 3 days. The antibodies were tested in two panels to study the effects of 6 drugs in 5 dilutions (1-10000 nM) (clofarabine, bortezomib, dexamethasone, navitoclax, venetoclax and omipalisib) on 11 cell populations, namely hematopoietic stem cells (HSCs) (CD34+CD38-), common progenitor cells (CPCs) (CD34+CD38+), monocytes (CD14+), B cells (CD45+CD19+), cytotoxic T cells (CD45+CD3+CD8+), T helper cells (CD45+CD3+CD4+), NK-T cells (CD45+CD3+CD56+), NK cells (CD45+CD56+CD3-), clonal plasma cells (CD138+CD38+), other plasma cells (CD138+CD38-) and granulocytes (CD45+, SSC++). Annexin-V and 7AAD were used to distinguish live cell populations from apoptotic and dead cells. After 1 h incubation with antibodies, the plates were read with the iQue Screener PLUS instrument (Intellicyt). Counts for each population were used to generate four parameter nonlinear regression fitted dose response curves with GraphPad Prism 7. Three samples were tested in duplicate to assess reproducibility.
Results
To decrease the complexity of the assay, we tested all antibodies under wash and no wash conditions, and found that results from both conditions were comparable. To minimize the amount of sample needed as well as maximize the number of drugs tested and cell populations that can be detected, we set up the assay in both 96- and 384-well plates. The assay was highly reproducible when samples were tested in replicate and was scalable to a 384-well format without compromising sensitivity to detect rare populations such as plasma cells. Due to the differentiation of immature cells to specialized cell types, the drug responses of specific populations tended to drift. HSCs (CD34+CD38-) were shown to be refractory to the tested drugs compared to CPCs characterized as (CD34+CD38+) and other cell types. Interestingly, the proteasome inhibitor bortezomib was cytotoxic to all cell populations except for CD138+CD38- plasma cells. Clofarabine, a nucleoside analog used to treat ALL, effectively targeted CPC, NK and B cells, while HSCs and plasma cells were resistant. The glucocorticoid and immunosuppressive drug dexamethasone specifically targeted B and NK cells compared to T cell populations (CD8+, CD4+), while NK-T cells were modestly sensitive. The cell population response patterns were similar in samples derived from MM, AML and healthy individuals, highlighting that the drug responses are highly cell type specific.
Summary
Using a high content, multi-parametric assay, we could rapidly assess the effect of several drugs on specific cell populations in individual patient samples. Our results demonstrate that many drugs preferentially affect different hematological cell lineages. Although heterogeneity was observed between individual patients, the pattern of cytotoxic response exhibited by specific cell types was consistent among samples derived from MM, AML and healthy donors. The assay will be useful to identify drugs with maximal on-target and minimal off-target specificity, and can potentially be used to guide treatment decision and predict patient response
Heckman:Celgene: Research Funding; Pfizer: Research Funding.
Background: Immunoglobulin light-chain (AL) amyloidosis is a rare disease caused by plasma cell secretion of misfolded light chains that assemble as amyloid fibrils and deposit on vital organs ...including the heart and kidneys, causing organ dysfunction. Plasma cell directed therapeutics, aimed at preferentially eliminating the clonal population of amyloidogenic cells in bone marrow are expected to reduce production of toxic light chain and alleviate deposition of amyloid thereby restoring healthy organ function.
Melphalan flufenamide ethyl ester, melflufen, is a peptidase potentiated alkylating agent with potent toxicity in myeloma cells. Melflufen is highly lipophilic, permitting rapid cellular uptake, and is subsequently enzymatically cleaved by aminopeptidases within cells resulting in augmented intracellular concentrations of toxic molecules, providing a more targeted and localized treatment. Previous data demonstrating multiple myeloma plasma cell sensitivity for melflufen suggests that the drug might be useful to directly eliminate amyloidogenic plasma cells, thereby reducing the amyloid load in patients. Furthermore, the increased intracellular concentrations of melflufen in myeloma cells indicates a potential reduction in systemic toxicity in patients, an important factor in the fragile amyloidosis patient population.
To assess potential efficacy in amyloidosis patients and to explore the mechanism of action, we examined effects of melflufen on amyloidogenic plasma cells in vitro and in vivo.
Methods: Cellular toxicity and apoptosis were measured in response to either melflufen or melphalan in multiple malignant human plasma cell lines, including the amyloidosis patient derived light chain secreting ALMC-1 and ALMC-2 cells, as well as primary bone marrow cells from AL amyloidosis patients, using annexin V and live/dead cell staining by multicolor flow cytometry, and measurement of cleaved caspases. Lambda light chain was measured in supernatant by ELISA, and intracellular levels were detected by flow cytometry. To assess efficacy of melflufen in vivo, the light chain secreting human myeloma cell line, JJN3, was transduced with luciferase and adoptively transferred into NSG mice. Cell death in response to melflufen or melphalan was measured by in vivo bioluminescence, and serum light chain was monitored.
Results: Melflufen demonstrated increased potency against multiple myeloma cell lines compared to melphalan, inducing malignant plasma cell death at lower doses on established light chain secreting plasma cell lines. While ALMC-1 cells were sensitive to both melphalan and melflufen, the IC50 for melphalan at 960 nM was approximately 3-fold higher than melflufen (334 nM). However, ALMC-2 cells were relatively insensitive to melphalan (12600 nM), but maintained a 100-fold increase in sensitivity to melflufen (121 nM). Furthermore, while 40% of primary CD138+ plasma cells from patients with diagnosed AL amyloidosis responded to melflufen treatment in vitro, only 20% responded to melphalan with consistently superior IC50 values for melflufen (Figure 1). Light chain secreting cell lines and AL amyloidosis patient samples were further analyzed by single cell sequencing.
We further examined differential effects on apoptosis and the unfolded protein response in vitro in response to either melflufen or melphalan. This is of particular interest in amyloidosis, where malignant antibody producing plasma cells possess an increased requirement for mechanisms to cope with the amplified load of unfolded protein and associated ER stress.
As AL amyloidosis is ultimately a disease mediated by secretion of toxic immunoglobulin, we assessed the effects of melflufen on the production of light chain in vitro, measuring a decrease in production of light chain in response to melflufen treatment. Finally, we took advantage of a recently described adoptive transfer mouse model of amyloidosis to assess the efficacy of melflufen and melphalan in eliminating amyloidogenic clones and reducing the levels of toxic serum light chain in vivo.
Conclusions: These findings provide evidence that melflufen mediated toxicity, previously described in myeloma cells, extends to amyloidogenic plasma cells and further affects the ability of these cells to produce and secrete toxic light chain. This data supports the rationale for the evaluation of melflufen in patients with AL amyloidosis.
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Flanagan:Oncopeptides AB: Employment. Slipicevic:Oncopeptides AB: Employment. Holstein:Celgene: Consultancy; Takeda: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; Genentech: Membership on an entity's Board of Directors or advisory committees; Sorrento: Consultancy. Lehmann:Oncopeptides AB: Employment. Nupponen:Oncopeptides AB: Employment. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding.
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