Riikka Karjalainen

Quantitative scoring of differential drug sensitivity for individually optimized anticancer therapies

We developed a systematic algorithmic solution for quantitative drug sensitivity scoring (DSS), based on continuous modeling and integration of multiple dose-response relationships in high-throughput compound testing studies. Mathematical model estimation and continuous interpolation makes the scoring approach robust against sources of technical variability and widely applicable to various experimental settings, both in cancer cell line models and primary patient-derived cells. Here, we demonstrate its improved performance over other response parameters especially in a leukemia patient case study, where differential DSS between patient and control cells enabled identification of both cancer-selective drugs and drug-sensitive patient sub-groups, as well as dynamic monitoring of the response patterns and oncogenic driver signals during cancer progression and relapse in individual patient cells ex vivo. An open-source and easily extendable implementation of the DSS calculation is made freely available to support its tailored application to translating drug sensitivity testing results into clinically actionable treatment options.

Female Mice Expressing Constitutively Active Mutants of FSH Receptor Present with a Phenotype of Premature Follicle Depletion and Estrogen Excess

Strong gain-of-function mutations have not been identified in humans in the FSH receptor (FSHR), whereas such mutations are common among many other G protein-coupled receptors. In order to predict consequences of such mutations on humans, we first identified constitutively activated mutants of the mouse (m) Fshr and then expressed them under the human anti-Müllerian hormone promoter in transgenic mice or created knock-in mutation into the mouse genome. We show here that mutations of Asp580 in the mFSHR significantly increase the basal receptor activity. D580H and D580Y mutations of mFSHR bind FSH, but the activity of the former is neither ligand-dependent nor promiscuous towards LH/human choriogonadotropin stimulation. Transgenic expression of mFshr(D580H) in granulosa cells leads to abnormal ovarian structure and function in the form of hemorrhagic cysts, accelerated loss of small follicles, augmented granulosa cell proliferation, increased estradiol biosynthesis, and occasional luteinized unruptured follicles or teratomas. The most affected mFshr(D580H) females are infertile with disturbed estrous cycle and decreased gonadotropin and increased prolactin levels. Increased estradiol and prolactin apparently underlie the enhanced development of the mammary glands, adenomatous pituitary growth, and lipofuscin accumulation in the adrenal gland. The influence of the mFSHR(D580Y) mutation is milder, mainly causing hemorrhagic cysts in transgenic mFSHR(D580Y) and mFSHR(D580Y) -knock-in mice. The results demonstrate that gain-of-function mutations of the FSHR in mice bring about distinct and clear changes in ovarian function, informative in the search of similar mutations in humans.

JAK1/2 and BCL2 inhibitors synergize to counteract bone marrow stromal cell-induced protection of AML

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.

Enhanced sensitivity to glucocorticoids in cytarabine-resistant AML.

We sought to identify drugs that could counteract cytarabine resistance in acute myeloid leukemia (AML) by generating eight resistant variants from MOLM-13 and SHI-1 AML cell lines by long-term drug treatment. These cells were compared with 66 ex vivo chemorefractory samples from cytarabine-treated AML patients. The models and patient cells were subjected to genomic and transcriptomic profiling and high-throughput testing with 250 emerging and clinical oncology compounds. Genomic profiling uncovered deletion of the deoxycytidine kinase (DCK) gene in both MOLM-13- and SHI-1-derived cytarabine-resistant variants and in an AML patient sample. Cytarabine-resistant SHI-1 variants and a subset of chemorefractory AML patient samples showed increased sensitivity to glucocorticoids that are often used in treatment of lymphoid leukemia but not AML. Paired samples taken from AML patients before treatment and at relapse also showed acquisition of glucocorticoid sensitivity. Enhanced glucocorticoid sensitivity was only seen in AML patient samples that were negative for the FLT3 mutation (P=0.0006). Our study shows that development of cytarabine resistance is associated with increased sensitivity to glucocorticoids in a subset of AML, suggesting a new therapeutic strategy that should be explored in a clinical trial of chemorefractory AML patients carrying wild-type FLT3.

Identification of the genes regulated by Wnt-4, a critical signal for commitment of the ovary

The indifferent mammalian embryonic gonad generates an ovary or testis, but the factors involved are still poorly known. The Wnt-4 signal represents one critical female determinant, since its absence leads to partial female-to-male sex reversal in mouse, but its signalling is as well implicated in the testis development. We used the Wnt-4 deficient mouse as a model to identify candidate gonadogenesis genes, and found that the Notum, Phlda2, Runx-1 and Msx1 genes are typical of the wild-type ovary and the Osr2, Dach2, Pitx2 and Tacr3 genes of the testis. Strikingly, the expression of these latter genes becomes reversed in the Wnt-4 knock-out ovary, suggesting a role in ovarian development. We identified the transcription factor Runx-1 as a Wnt-4 signalling target gene, since it is expressed in the ovary and is reduced upon Wnt-4 knock-out. Consistent with this, introduction of the Wnt-4 signal into early ovary cells ex vivo induces Runx-1 expression, while conversely Wnt-4 expression is down-regulated in the absence of Runx-1. We conclude that the Runx-1 gene can be a Wnt-4 signalling target, and that Runx-1 and Wnt-4 are mutually interdependent in their expression. The changes in gene expression due to the absence of Wnt-4 in gonads reflect the sexually dimorphic role of this signal and its complex gene network in mammalian gonad development.

Statistical detection of quantitative protein biomarkers provides insights into signaling networks deregulated in acute myeloid leukemia

The increasing coverage and sensitivity of LC‐MS/MS‐based proteomics have expanded its applications in systems medicine. In particular, label‐free quantitation approaches are enabling biomarker discovery in terms of statistical comparison of proteomic profiles across large numbers of clinical samples. However, it still remains poorly understood how much protein markers can add novel insights compared to markers derived from mRNA transcriptomic profiling. Using paired label‐free LC‐MS/MS and gene expression microarray measurements from primary samples of patients with acute myeloid leukemia (AML), we demonstrate here that while the quantitative proteomic and transcriptomic profiles were highly correlated, in general, the marker panels showing statistically significant expression changes across the disease and healthy groups were profoundly different between protein and mRNA levels. In particular, the proteomic assay enabled unique links to known leukemic processes, which were missed when using the transcriptomic profiling alone, as well as identified additional links to metabolic regulators and chromatin remodelers, such as GPX1, fumarate hydratase, and SET oncogene, which have subsequently been evaluated in independent AML samples. Overall, these results highlighted the complementary and informative view obtained from the quantitative LC‐MS/MS approach into the AML deregulated signaling networks.

Identification of precision treatment strategies for relapsed/ refractory multiple myeloma by functional drug sensitivity testing

Novel agents have increased survival of multiple myeloma (MM) patients, however high-risk and relapsed/refractory patients remain challenging to treat and their outcome is poor. To identify novel therapies and aid treatment selection for MM, we assessed the ex vivo sensitivity of 50 MM patient samples to 308 approved and investigational drugs. With the results we i) classified patients based on their ex vivo drug response profile; ii) identified and matched potential drug candidates to recurrent cytogenetic alterations; and iii) correlated ex vivo drug sensitivity to patient outcome. Based on their drug sensitivity profiles, MM patients were stratified into four distinct subgroups with varied survival outcomes. Patients with progressive disease and poor survival clustered in a drug response group exhibiting high sensitivity to signal transduction inhibitors. Del(17p) positive samples were resistant to most drugs tested with the exception of histone deacetylase and BCL2 inhibitors. Samples positive for t(4;14) were highly sensitive to immunomodulatory drugs, proteasome inhibitors and several targeted drugs. Three patients treated based on the ex vivo results showed good response to the selected treatments. Our results demonstrate that ex vivo drug testing may potentially be applied to optimize treatment selection and achieve therapeutic benefit for relapsed/refractory MM.Novel agents have increased survival of multiple myeloma (MM) patients, however high-risk and relapsed/refractory patients remain challenging to treat and their outcome is poor. To identify novel therapies and aid treatment selection for MM, we assessed the ex vivo sensitivity of 50 MM patient samples to 308 approved and investigational drugs. With the results we i) classified patients based on their ex vivo drug response profile; ii) identified and matched potential drug candidates to recurrent cytogenetic alterations; and iii) correlated ex vivo drug sensitivity to patient outcome. Based on their drug sensitivity profiles, MM patients were stratified into four distinct subgroups with varied survival outcomes. Patients with progressive disease and poor survival clustered in a drug response group exhibiting high sensitivity to signal transduction inhibitors. Del(17p) positive samples were resistant to most drugs tested with the exception of histone deacetylase and BCL2 inhibitors. Samples positive for t(4;14) were highly sensitive to immunomodulatory drugs, proteasome inhibitors and several targeted drugs. Three patients treated based on the ex vivo results showed good response to the selected treatments. Our results demonstrate that ex vivo drug testing may potentially be applied to optimize treatment selection and achieve therapeutic benefit for relapsed/refractory MM.

Abstract 3175: Genomic and transcriptomic data integration in chronic myelomonocytic leukemia reveals a novel fusion gene involving onco-miR-125b-2

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Chronic myelomonocytic leukemia (CMML) is a rare malignancy characterized by increased peripheral monocytosis and dysplasia in a single- or multilineage fashion. Gene mutations so far reported in CMML include TET2, CBL, NRAS, KRAS, RUNX1 and EZH2 but their pathogenic role and driver status in the disease remains unclear. Altered expression of the microRNA miR-125b has been implicated in the pathogenesis of many types of cancers, including myeloid leukemias and Down syndrome-associated acute myeloid leukemia (DS-AML). In addition, this miRNA has been shown to play an important role in hematopoiesis and the regulation of immune cell response. Here, integration of data from next-generation transcriptome sequencing, exome sequencing and array-CGH in a CMML patient (trisomy 21 by cytogenetics) led to the identification of a novel gene fusion event involving the nuclear receptor interacting protein NRIP1 gene and the open reading frame C21orf34 (both at 21q21 approximately 1 MB apart). The fusion was validated by capillary sequencing and found to involve two copy number transition breaks, inversion of the intervening region and the upregulation of the 3′ end of C21orf34. This intronic region harbors a cluster of three miRNAs: miR-let7c, miR-99a, and miR-125b-2. Based on genomic breakpoint analysis, the gene fusion took place just upstream of miR-125b-2. Consistent with this, only miR-125b-2 was highly expressed in the sample, and was processed to a mature miRNA. By RT-PCR, increased expression of miR-125b-2 was also observed in four other CMML patients and five CML patients when compared to healthy bone marrow controls. In contrast, five AML cases studied showed expression levels similar to or lower than that of controls. Interestingly, one AML patient with trisomy 21 had very high levels of miR-125b-2. We found the NRIP1-C21orf34 fusion only in our index patient and therefore other mechanisms of miRNA deregulation at 21q21 in CMML/CML and AML+21 will also exist. In conclusion, we describe for the first time a fusion gene involving miR-125b-2 in CMML, a previously recognized and well-studied onco-miR, which is known to impact on self-renewal of hematopoietic cell precursors. We also detected overexpression of miR-125b-2 in all CMML samples studied suggesting a key pathogenetic driver gene role for this micro-RNA. The assessment of miR-125b-2 levels could potentially be applied to the diagnosis and follow-up of patients with CMML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3175. doi:1538-7445.AM2012-3175

Abstract 5067: Exome sequencing reveals both DNA sequence and copy number changes in AML: Potential driver changes and mechanisms of drug resistance revealed from serial samples from the same patients

Despite significant advances in characterizing the molecular genetics of AML, the clonal evolution of leukemic cells and the dynamic impact of genomic changes on the development of the disease and progression to drug resistance are not well understood. Here, we applied next-generation sequencing to quantify aberrant tumor subclones carrying specific mutant alleles of key cancer genes and developed a method to extract quantitative high-resolution copy number changes across the genome using exome sequencing data from matching cancer and normal DNA. Serial bone marrow (BM) samples collected from diagnosis to relapse to post-treatment drug resistance in a patient-centric manner made it possible to trace the clonal evolution of AML and to identify variants potentially involved in drug resistance. Exome sequencing from AML blast cells and normal skin biopsies was performed as part of the Finnish Hematology Registry and Biobanking (FHRB) effort. Consecutive paired samples from different patients revealed unique genetic patterns of clonal evolution and cancer progression in each patient. In a pre-resistant sample of one AML M5 patient, we identified four closely spaced insertions in the Wilm9s Tumor (WT1) suppressor gene, none of which appear on the same sequence reads. This suggests the presence of multiple distinct leukemic subclones even before treatment resistance and underscores the strong selective advantage conferred by WT1 mutations. After relapse, one of the subclones was lost, and another one significantly increased suggesting that the relapse arose from the expansion of a pre-existing resistant subclone. In this patient, recurrent clones otherwise featured similar copy number changes and the same fusion genes as the primary diagnostic sample. In another AML patient developing recurrence an opposite pattern was observed: The relapsed, drug-resistant cells displayed an enormous increase of small microdeletions compared to the diagnostic, pre-treatment sample, while almost all sequence-level alterations in potential cancer genes were the same between the two samples. This suggests that a distinct type of DNA repair deficiency may have contributed to the drug resistant clone in this patient. Conclusions: Exome sequencing from paired samples of AML cells before and after relapse makes it possible to trace the clonal evolution of the disease and study the impact of therapy both at the level of sequence alterations of key cancer genes and simultaneously at the level of copy number changes inferred from exome sequence data. This analysis has highlighted multiple genomic patterns by which resistance may evolve in vivo during cancer treatment. Refined bioinformatic analysis and interpretation of exome-seq data provides a rich resource to identify genetic biomarkers of drug response and minimal residual disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5067. doi:1538-7445.AM2012-5067

Abstract 5588: Functional drug sensitivity and resistance profiling of AML patient cells defines a disease-specific combination of druggable signal addictions.

Numerous underlying molecular events have been described in acute myeloid leukemia (AML), but still, the fundamental disease mechanisms are poorly understood. Several targeted therapies have been investigated for improved AML therapy but have not succeeded to date, likely due to the inability to identify the patient subgroups that are most likely to benefit. Here, we describe functional profiling of AML patient cells ex vivo with a drug sensitivity and resistance (DSRT) platform in order to distinguish disease- and patient-specific molecular vulnerabilities and individualized therapeutic strategies. The oncology drug collection covers 306 anti-cancer agents including 131 approved, 107 investigational and 68 experimental compounds. Each compound is tested in a dose response series allowing for calculation of drug sensitivity scores. The functional exploration of AML patient samples was accompanied with comprehensive molecular profiling and clinical background data to link drug sensitivities to molecular aberrations and predictive biomarkers. Comparison of the drug sensitivity profiles of 24 AML patient and 5 control samples revealed that targeted inhibitors often exhibit no to little effect in the controls and the majority of AML patient samples with only a subset of patients showing very selective responses, indicating that cancer specific vulnerabilities can be detected with the DSRT platform. Clustering of the drug responses among patient and control samples identified distinct subgroups of patients and drugs. Each of these groups could be defined by a separate drug class, implying that the linked samples were addicted to the corresponding signaling networks. Specifically, we saw select addictions to drug classes such as dasatinib/VEGFR TKIs, MEK inhibitors, rapalogs, JAK inhibitors, mTOR inhibitors in 6 (25 %), 5 (21%), 5 (21%), 5 (21%), and 6 (25%) of the patients, respectively. Together, these sensitivities highlight a set of disease-specific and individualized signaling nodes that can serve as starting points for personalized combination therapy strategies in AML. Further, integration of DSRT results with molecular and clinical profiling link some of the selective drug responses to clinically actionable markers. For example, dasatinib sensitivity was found to be predominant in AML M5 patients, suggesting that this drug is a promising therapeutic candidate for relapsed and refractory AML M5 patients. In conclusion, the DSRT platform can be used as a functional interrogation of patient cells leading to the identification of druggable vulnerabilities that can be used for personalized medicine strategies. Furthermore, insight into disease-specific signal addictions and establishment of how these are linked can aid in deconvoluting the complex molecular disease mechanisms of cancers and generate strategies for novel drug development. Citation Format: Tea Pemovska, Bhagwan Yadav, Riikka Karjalainen, Evgeny Kulesskiy, Mika Kontro, Muntasir Mamun Majumder, Laura Turunen, Ida Lindenschmidt, Anna Lehto, Jonathan Knowles, Caroline Heckman, Kimmo Porkka, Tero Aittokallio, Olli Kallioniemi, Krister Wennerberg. Functional drug sensitivity and resistance profiling of AML patient cells defines a disease-specific combination of druggable signal addictions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5588. doi:10.1158/1538-7445.AM2013-5588