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In this issue of Blood , Ghia et al observe that a proportion of patients with chronic lymphocytic leukemia (CLL) harbor mutations in genes involved in the Hedgehog (Hh) pathway resulting in expression of glioma-associated oncogene homolog 1 (GLI1). They also find that GLI1 is expressed in CLL cells without evidence of Hh mutations and that the high GLI1 levels correlate with disease progression. 1

Recent advances in understanding the major bottlenecks in derivation of engraftable HSCs and lymphoid cells from pluripotent stem cells (PSC), have identified deficiencies in NOTCH and HOXA signaling as contributing factors to the observed functional deficits of PSC-derived hematopoietic progenitors. However, little is known about the mechanisms that are essential for establishing these pathways during PSC differentiation. Here, we revealed the critical role of SOX17 in linking HOXA and NOTCH-mediated arterial programs in hemogenic endothelium (HE) and specification of definitive lympho-myeloid hematopoiesis. Using SOX17-knockout (SOX17-/-) and SOX17 DOX-inducible (iSOX17) hESCs, we found that SOX17-deficiency substantially reduces formation of CD144+CD43-CD73-DLL4+CXCR4+/- arterial HE and definitive lympho-myeloid hematopoiesis, while SOX17 upregulation at mesodermal stage of development causes the opposite effect. Molecular profiling of HE generated from iSOX17 hESCs in DOX+ and DOX- conditions using RNAseq, SOX17 ChIPseq and ATACseq, revealed that SOX17 overexpression upregulates 522 genes enriched in NOTCH, TGFb, HEDGEHOG and WNT signaling, including DLL1, DLL4, NOTCH4, LFNG, WNT5a, WNT5b, GLI3, and genes associated with HSC development, CXCR4,KITLG and ALDH1A2. In addition, we noted significant upregulation of HOXA7,HOXA9, HOXA10, HOXB8, HOXC4 and CDX2 homeobox genes in SOX17-induced cultures, with no expression of HOXA genes observed in HE from SOX17-/- cells. ChIPSeq analysis revealed DOX+ specific SOX17 binding at transcriptional start sites (TSS) of 316 significantly upregulated genes, including ALDH1A2, CDX2, DLL1, DLL4, HEY1, HOXA7, HOXB8, HOXC4 and KITLG, suggesting that upregulation of these genes could be explained by their direct activation by SOX17. Since ALDH1A2 and CDX2 are known to play a role in the activation of HOXA genes, we investigated whether SOX17's effect on HOXA expression could also be mediated by ALDH1A2 and CDX2. We found that adding ALDH1 inhibitor to DOX+ cultures had no effect on arterial HE development and HOXA expression. In contrast, transfection of iSOX17 hPSCs cultures with CDX2 shRNA significantly decreased arterial HE formation and downregulated HOXA7, HOXA9, and HOXA10 expression. Overall, our studies indicate that SOX17 plays a critical role in the activation and integration of arterial and HOXA programs in HE, which is mediated by CDX2. These findings will be important for designing a strategy for direct HSC fate programming from hPSCs. Disclosures Uenishi: Casebia Therapeutics: Employment. Slukvin:Cynata Therapeutics: Consultancy, Other: Founder and Stockholder.

Programs that control early lineage fate decisions and transitions from embryonic to adult human cell types during development are poorly understood. Using human pluripotent stem cells (hPSCs), in the present study, we reveal reduction of Hedgehog (Hh) signaling correlates to developmental progression of hematopoiesis throughout human ontogeny. Both chemical- and gene-targeting–mediated inactivation of Hh signaling augmented hematopoietic fate and initiated transitions from embryonic to adult hematopoiesis, as measured by globin regulation in hPSCs. Inhibition of the Hh pathway resulted in truncation of Gli3 to its repressor, Gli3R, and was shown to be necessary and sufficient for initiating this transition. Our results reveal an unprecedented role for Hh signaling in the regulation of adult hematopoietic specification, thereby demonstrating the ability to modulate the default embryonic programs of hPSCs.

Background : although azacitidine (AZA) is an established frontline therapy for HR-MDS, approximately 40% of the patients fail to respond and virtually all of the responders will ultimately relapse. We previously reported the poor outcome of patients who failed AZA treatment for whom a median survival of 6 months is expected (Prebet, J Clin Oncol. 2011;29(24):3322). Overexpression of SMO a member of the hedgehog pathways has been observed in HR-MDS. Preclinical data have shown the activity of hedgehog pathway inhibitors and suggested synergism with Hypomethylating agents (HMA) (Zou, PLoSONE 2015; 10(8):e0136843). To harness potential synergism, we designed an add-on trial in which sonidegib (LDE-225), an oral inhibitor, was added to AZA in patients who failed AZA monotherapy. Patients and methods: Eligible patients should have IPSS Int-2 or HR-MDS or AML, (to have 10% to 30% marrow blasts), have received a minimum of 6 cycles of AZA monotherapy and to have failed to achieve a response to AZA (defined as CR, CRi, PR or HI as per IWG criteria) in the absence of marrow progression or have relapsed after achieving a response. Patients were scheduled to receive sonidegib daily from day 1 to 28 of 28-day cycles while continuing on AZA at the maximum previously tolerated dose (ie 50 to 75 mg/m²/d for 5-7 days). Two sonidegib dose levels were evaluated in the phase 1 part of the trial (400 and 800 mg/d) according to a classical 3+3 scheme. Results: 23 patients, median age 76, were included in the study. One withdrew consent before starting the study treatment and is not included in the analysis. Characteristics of the remaining 22 patients are summarized in the Table. At the 800 mg/d dose level, 3 pts experienced DLT (grade 4 ASAT elevation (1 pt); grade 4 CPK elevation (2 pts)). The recommended dose for the expansion phase was 400 mg/d and no DLT was observed. 19 patients received 400 mg/d. Pts received a median of 3.5 cycles [IQR 2 ; 5], and 5 pts (23 %) received ≥ 6 cycles. All of them were withdrawn from study: 5 (26%) for disease progression (including 1 pt who progressed before starting cycle 1 and did not received treatment), 5 (26%) for death (all due to infection in the context of active disease) and 9 for miscellaneous reasons. 25 SAEs were reported including 23 infections, 1 intra cranial hemorrhage and 1 urinary retention. The most frequent non-hematologic toxicity was infection that occurred in 16/22 (73%) pts. In addition, 243 non hematologic toxicities of any grade were recorded in 20 pts for a total of 83 cycles, all but 3 of grade 1-2, including GI, cutaneous and musculo-skeletal toxicities and fatigue. Hospitalization was required in 45% of the pts. Only 3 responses were observed (1 CR, 1 marrow CR and 1 PR). Response durations were 1, 9 and 15 months .With a median follow-up of 23 months, 13 pts have died and the median overall survival is 6.8 months. Conclusion Sonidegib at the dose of 400 mg/d can be given in combination with azacitidine with an acceptable toxicity. With a response rate of only 13%, our results indicate that the addition of sonidegib failed to reverse resistance to azacitidine in pts with higher risk MDS. This contrasts with the positive effect of the combination of glasdegib, another hedgehog pathway inhibitor with cytarabine in pts with AML and MDS (Cortes, Blood 2016 128:99). Besides potential differences in drug potency, our results may highlight the difficulty to identify active agents in the difficult-to-treat patient population selected after HMA therapy failure. The recommended dose-schedule of AZA-sonidegib established here could be used for further investigation in previously untreated patients. Table. Table. Disclosures No relevant conflicts of interest to declare.

Leukemia is a developmental disease of the hematopoietic system. In this issue of Blood, Dagklis et al1 identified hyperactivation of the hedgehog developmental pathway in a subgroup of T-cell acute lymphoblastic leukemias (T-ALLs). Given the availability of US Food and Drug Administration (FDA)–approved hedgehog-inhibiting drugs and the extremely poor prognosis of relapsed T-ALL, this discovery may present a new therapeutic opportunity.

Background: Acute myeloid leukemia (AML) is initiated and maintained by a subpopulation of leukemic stem cells (LSCs), which are defined by their property to serially engraft immunocompromised mice. Based on this a LSC-associated gene expression was identified which was shown to be prognostically relevant in a retrospective analysis (Eppert et al, Nature Medicine 2011). microRNAs (miRs) play important roles in leukemogenesis, serve as potential tumor markers and novel therapeutic targets. In the current analysis we wanted to establish a miRNA profile of functionally validated LSCs from AML patients of various molecular and cytogenetic subgroups by conducting a comprehensive functional genomics study. Method: In total, 17 de novo AML samples were sorted for CD34+ granulocyte-macrophage progenitor (GMP), CD34+ lymphoid-primed multi-potential progenitor (LMPP) and CD34- subpopulations. Sorted cells were transplanted into sublethally irradiated non-obese diabetic/severe combined immunodeficient Gamma (NSG) mice. Those subpopulations that gave rise to human leukemic cell engraftment > 1% were classified as LSCs and sub-divided into LMPP-LSCs and GMP-LSCs and compared to the CD34- cell compartment which did not give rise to human leukemic cell engraftment (non-LSC). In parallel corresponding counterparts were highly purified from healthy bone marrow (BM). miRNA libraries were prepared using the Illumina TruSeqTM Small RNA Sample Preparation Kit and sequenced as single end reads on a Illumina HiSeq 2000. The bioconductor package limma was used for identifying differentially expressed miRNAs, the made4 package for correspondence analysis and between group comparisons. Quantitative RT-PCR validation on bulk and sorted populations (LMPP-LSC, GMP-LSC, CD34-) tested the expression levels of the identified miRNAs. Results: We assessed the engraftment potential of 17 AML samples, sorted into the LMPP, GMP and CD34- fraction. The LMPP or GMP fractions of 8 patients showed a leukemic graft in the NSG mouse model, whereas the CD34- fractions were not able to show a human engraftment in these mice. To the end subpopulations of 8 validated patient samples and corresponding stem-, progenitor, and CD34- subpopulations of normal bone marrow samples (n=3) were analyzed. Global miRNA expression profiles of subpopulations from normal and patient samples were compared using correspondence analysis based between group analysis. First, LSC subpopulations were compared to the leukemic CD34- Non-LSCs: between the LMPP-LSCs and the CD34- non-LSCs 15 miRNAs were differentially expressed in the LMPP-LSCs; between the GMP-LSCs and the CD34- non-LSCs only six miRNAs were found to be differentially expressed. Using these miRNAs, an unsupervised hierarchical clustering showed that the LMPP-LSCs, the GMP-LSCs and leukemic CD34- samples formed non-overlapping clusters. In normal bone marrow, 12 miRs showed differential expression between normal BM LMPP and CD34- population and 36 miRNAs between BM GMP vs. BM CD34- cells. We then compared the miRs that were differentially expressed in the patients compared to the differentially expressed miRs between the normal BM populations: except for one miR (miR-182), the fifteen differentially expressed miRs in the LMPP-LSC versus leukemic CD34- samples did not show overlap with the twelve miR significantly differentially expressed between normal BM LMPP and CD34- cells. When the GMP-LSC to leukemic CD34- Non-LSC comparison was cross-compared to the BM GMP to normal BM CD34- comparison, only two miRs (miR-148a, miR-320a) were unique to the patient samples. When we finally compared the LMPP-LSC specific 14 miR to the 2 GMP-LSC specific miRs, interestingly, we observed that the GMP-LSC miRs were a subset of the LMPP-miRs. Thus, 14 miRs defined a set of miRs differentially expressed in functionally validated AML LSCs. Importantly, 4 of these 14 miRs represent miR-941 isoforms. This miR has not been linked to leukemogenesis yet. It is one of the very few human-specific miRs which developed de novo in the human lineage after separation of the human and the chimpanzee lineages between six and one million years ago and preferentially targets genes in hedgehog- and insulin-signalling pathways. Taken together, combining functional validation of AML LSCs with next generation sequencing allows to identify miRs so far not linked to AML biology and LSC properties. Figure Figure. Disclosures Mulaw: NuGEN: Honoraria. Buske:Celltrion, Inc.: Consultancy, Honoraria.